Administering methylation-controlled J protein (MCJ) SIRNA to a kidney cell

ABSTRACT

The invention relates, in part, to methods and compositions that are useful to modulate metabolic function of cells in vivo or in vitro. In some aspects the invention includes methods and/or compositions that increase metabolism in cells, tissues, organs, and/or subjects. In certain aspects the invention includes methods and/or compositions useful to decrease metabolism in cells, tissues, organs, and/or in subjects.

RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.15/805,534, filed Nov. 7, 2017, which is a continuation of U.S.application Ser. No. 14/413,927, filed Jan. 9, 2015, which issued Mar.10, 2020 as U.S. Pat. No. 10,583,169, which is a National Stage Filingunder U.S.C. § 371 of PCT International Application PCT/US2013/49885,filed Jul. 10, 2013 which was published under PCT Article 21(2) inEnglish, which claims benefit under 35 U.S.C. § 119(e) of U.S.Provisional Application No. 61/670,345, filed Jul. 11, 2012 and thecontent of each of which is incorporated by reference herein in itsentirety.

GOVERNMENT INTEREST

This invention was made with government support under R21 CA127099awarded by the National Institutes of Health. The government has certainrights in the invention.

FIELD OF THE INVENTION

The invention relates, in part, to methods and compounds that are usefulto regulate metabolic functions.

BACKGROUND

Co-chaperones modulate the activity of chaperones, primarily heat shockproteins (HSPs), either directly or indirectly through the recruitmentof other proteins (Walsh et al., 2004, EMBO Rep 5, 567-571). Althoughthe regulation and role of chaperones have been extensively studied,little is known about the function of the large number of identifiedco-chaperones. The DnaJ family of co-chaperones is the largest and themost diverse with 49 identified members in humans. It is characterizedby the presence of the highly conserved 70 aa J-domain containing aHis-Pro-Asp motif that binds to the ATPase domain of HSP70 familymembers and promotes their ATPase activity (Walsh et al., 2004, EMBO Rep5, 567-571; Mitra et al., 2009, Clin Exp Metastasis 26, 559-567;Sterrenberg et al., 2011, Cancer Lett 312, 129-142). The members of thisfamily have been classified in three subfamilies according to thepresence and nature of sequences other than the J-domain (DnaJA, DnaJBand DnaJC subfamilies) (Kampinga et al., 2009, Cell Stress Chaperones14, 105-111). The DnaJA subfamily contains a Gly/Phe (G/F) rich regionand a Cys repeat region, while the DnaJB subfamily contains the G/Fregion but lacks the Cys-repeat region. DnaJC subfamily members arehighly diverse. They lack both the G/F and Cys-repeat regions, whiletheir J domain can be located at any position in the protein. However,most DnaJC members have less characterized non-classical domains thatseem to provide specificity for partner binding and function.

DnaJ co-chaperones can interact with proteins other than HSPs in aDnaJ-domain independent manner to mediate specific functions(Sterrenberg et al., 2011 Cancer Lett 312, 129-142). However, only a fewtargets have been identified. Within the mitochondria, the DNApolymerase gamma (Polga) has been shown to interact with Tid1 (Hayashiet al., 2006 Nat Med 12, 128-132), and more recent studies have alsoidentified p53 as another potential target (Ahn et al., 2010 Oncogene29, 1155-1166). DnaJA1 has also been recently found to interact withAdenosin Induced Deaminase (AID) in B cells and modulate its activityduring class switching recombination (Orthwein et al., EMBO J 2011 Nov.15; 31(3):679-91). The role of DnaJ co-chaperones in cancer is afunctional aspect of these proteins that has begun to be investigated,although primarily in cell lines (Mitra et al., 2009, Clin ExpMetastasis 26, 559-567). In addition, only a few orthologs of human DnaJproteins have been identified in mouse (e.g., DnaJC10/ERdj5, DnaJB6/Mrjand DnaJA3/Tid1, DnaJA1/DjA1) (Terada et al., 2005, EMBO J 24, 611-622;Hosoda et al., 2010, Biochem J 425, 117-125; Lo et al., 2004, Mol CellBiol 24, 2226-2236; Hunter et al., 1999, Development 126, 1247-1258).

The absence of known domains and the presence of poorly characterizednon-classical domains have made it difficult to characterize thefunctions of most DnaJC family members. As a consequence, and despitethe large number of DnaJ co-chaperones identified in humans, theirfunction in normal and disease conditions remain mostly unknown.

SUMMARY OF THE INVENTION

According to one aspect of the invention, methods for treating ametabolic disease or condition in a subject are provided. The methodsinclude administering to a subject in need of such treatment anMCJ-modulating compound in an amount effective to treat the metabolicdisease or condition in the subject. In some embodiments, theMCJ-modulating compound decreases an MCJ polypeptide activity in thesubject and increases a metabolic activity of a mitochondrion in thesubject. In some embodiments, decreasing the MCJ polypeptide activitycomprises decreasing an MCJ polypeptide level or function. In someembodiments, the MCJ-modulating compound increases an MCJ polypeptideactivity in the subject and decreases a metabolic activity of amitochondrion in the subject. In some embodiments, increasing the MCJpolypeptide activity comprises increasing an MCJ polypeptide level orfunction. In certain embodiments, the method also includes reducing alevel of caloric intake of the subject. In some embodiments, the levelof caloric intake is reduced prior to, concurrent with, and/or afteradministration of the MCJ-modulating compound to the subject. In someembodiments, the metabolic disease or condition is overweight, weightgain, obesity, non-alcoholic fatty liver disease, diabetes,insulin-resistance, alcoholic fatty liver disease, dyslipidemia,steatosis (e.g., liver steatosis, heart steatosis, kidney steatosis,muscle steatosis), abeta-lipoproteinemia, glycogen storage disease,Weber-Christian disease, lipodystrophy; a liver disease, liverinflammation, hepatitis, steatohepatitis, Hepatitis C, Genotype 3Hepatitis C, Alpha 1-antitrypsin deficiency, acute fatty liver ofpregnancy, Wilson disease; a kidney disease; a heart disease,hypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV; aneurodegenerative disease, Parkinson's disease, Alzheimer's disease;cancer, or physical exercise. In some embodiments of the invention, themetabolic disease or condition is high cholesterol and reducing MCJactivity may be used in methods to reduce cholesterol in a cell, tissue,or subject. In certain embodiments, the metabolic disease or conditionis an eating disorder, anorexia, starvation, malnutrition, totalparenteral nutrition, severe weight loss, underweight, re-feedingsyndrome; gastrointestinal surgery-mediated metabolic alterations,jejuno-ilial bypass, gastric bypass; and inflammatory/infectiousconditions, jujunal diverticulosis with bacterial overgrowth, andinflammatory bowel disease. In some embodiments, the subject is afasting subject. In some embodiments, treating the metabolic disease orcondition comprises enhancing or inhibiting cytotoxic T cell activity.In some embodiments, the MCJ-modulating compound is administered in apharmaceutical composition. In certain embodiments, the pharmaceuticalcomposition further comprises a pharmaceutically acceptable carrier. Insome embodiments, the pharmaceutical composition further comprises atargeting agent. In some embodiments, the targeting agent is amitochondrial targeting agent. In some embodiments, the MCJ-modulatingcompound comprises an MCJ molecule, an anti-MCJ polypeptide antibody orfunctional fragment thereof, a small molecule MCJ inhibitor, or a smallmolecule MCJ enhancer. In certain embodiments, the MCJ molecule is anMCJ polypeptide or a nucleic acid that encodes an MCJ polypeptide. Insome embodiments, the subject does not have cancer. In some embodiments,the metabolic disease or condition is not cancer. In certainembodiments, the method also includes increasing a level of caloricintake of the subject. In some embodiments, the level of caloric intakeis increased prior to, concurrent with, and/or after administration ofthe MCJ-modulating compound to the subject.

According to another aspect of the invention, methods of alteringmitochondrial metabolism in a cell are provided. The methods includecontacting the cell with an exogenous MCJ-modulating compound in anamount effective to alter mitochondrial metabolism in the cell, whereinan MCJ-modulating compound that increases MCJ polypeptide activitydecreases mitochondrial metabolism in the cell and an MCJ-modulatingcompound that decreases MCJ polypeptide activity increases mitochondrialmetabolism in the cell. In some embodiments, increasing the MCJpolypeptide activity includes increasing a level or function of MCJpolypeptide in the cell. In certain embodiments, decreasing the MCJpolypeptide activity includes decreasing a level or function of MCJpolypeptide in the cell. In some embodiments, mitochondrial metabolismincludes mitochondrial respiration. In some embodiments, the cell is invitro. In some embodiments, the cell is in vivo. In certain embodiments,the cell is in a subject and the contacting includes administering theMCJ-modulating compound to the subject. In some embodiments, the subjectis a fasting subject. In some embodiments, the subject is a subject withan increased level of caloric intake. In certain embodiments, theMCJ-modulating compound is administered in a pharmaceutical composition.In some embodiments, the pharmaceutical composition also includes apharmaceutically acceptable carrier. In some embodiments, thepharmaceutical composition further comprises a targeting agent. In someembodiments, the targeting agent is a mitochondrial targeting agent. Incertain embodiments, the MCJ-modulating compound includes an MCJmolecule, an anti-MCJ polypeptide antibody or functional fragmentthereof, a small molecule MCJ inhibitor, or a small molecule MCJenhancer. In some embodiments, the MCJ molecule is an MCJ polypeptide ora nucleic acid that encodes an MCJ polypeptide. In some embodiments, theMCJ-modulating compound includes a mitochondrial targeting agent. Incertain embodiments, the subject has a metabolic disease or condition.In some embodiments, the metabolic disease or condition is overweight,weight gain, obesity, non-alcoholic fatty liver disease, diabetes,insulin-resistance, alcoholic fatty liver disease, dyslipidemia,steatosis (e.g., liver steatosis, heart steatosis, kidney steatosis,muscle steatosis), abeta-lipoproteinemia, glycogen storage disease,Weber-Christian disease, lipodystrophy; a liver disease, liverinflammation, hepatitis, steatohepatitis, Hepatitis C, Genotype 3Hepatitis C, Alpha 1-antitrypsin deficiency, acute fatty liver ofpregnancy, Wilson disease; a kidney disease; a heart disease,hypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV; aneurodegenerative disease, Parkinson's disease, Alzheimer's disease;cancer, or physical exercise. In some embodiments of the invention, themetabolic disease or condition is high cholesterol and reducing MCJactivity may be used in methods to reduce cholesterol in a cell, tissue,or subject. In some embodiments, the metabolic disease or condition isan eating disorder, anorexia, starvation, malnutrition, total parenteralnutrition, severe weight loss, underweight, re-feeding syndrome;gastrointestinal surgery-mediated metabolic alterations, jejuno-ilialbypass, gastric bypass; an inflammatory/infectious condition, jujunaldiverticulosis with bacterial overgrowth, or inflammatory bowel disease.In certain embodiments, the subject does not have cancer. In someembodiments, the metabolic disease or condition is not cancer.

According to another aspect of the invention, methods of modulatingcytotoxic T (CD8⁺ T) cell function in a subject are provided. Themethods include administering to a subject in need of such treatment anMCJ-modulating compound in an amount effective to increase or decreasean activity of an MCJ polypeptide in a CD8⁺ T cell, wherein an increasein MCJ polypeptide activity increases depolarization of a mitochondrionin the CD8⁺ T cell and a decrease in MCJ polypeptide activity decreasesdepolarization of a mitochondrion in the CD8⁺ T cell, and wherein theincrease or decrease in depolarization of the mitochondrion modulatescytotoxic T cell function in the subject. In some embodiments,increasing the activity of the MCJ polypeptide includes increasing thelevel or function of the MCJ polypeptide in the cell. In certainembodiments, decreasing the activity of the MCJ polypeptide includesdecreasing the level or function of the MCJ polypeptide in the cell. Insome embodiments, the MCJ-modulating compound is administered in apharmaceutical composition. In some embodiments, the pharmaceuticalcomposition also includes a pharmaceutically acceptable carrier. Incertain embodiments, the pharmaceutical composition also includes atargeting agent. In some embodiments, the targeting agent is amitochondrial targeting agent. In some embodiments, the MCJ-modulatingcompound includes an MCJ molecule, an anti-MCJ polypeptide antibody orfunctional fragment thereof, a small molecule MCJ inhibitor, or a smallmolecule MCJ enhancer. In some embodiments, the MCJ molecule is an MCJpolypeptide or a nucleic acid that encodes an MCJ polypeptide. Incertain embodiments, the subject does not have cancer.

According to another aspect of the invention, compositions that includean MCJ-modulating compound and a pharmaceutically acceptable carrier areprovided. In some embodiments, the composition also includes amitochondrial-targeting agent. In some embodiments, the MCJ-modulatingcompound includes an MCJ molecule, an anti-MCJ polypeptide antibody orfunctional fragment thereof, a small molecule MCJ inhibitor, or a smallmolecule MCJ enhancer. In some embodiments, the MCJ molecule is an MCJpolypeptide or a nucleic acid that encodes an MCJ polypeptide.

According to yet another aspect of the invention, methods of determiningthe presence or absence of a metabolic disease or condition in a subjectare provided. The methods include obtaining a biological sample from asubject; measuring a level of a MCJ molecule in the biological sample;and comparing the measured level with a control level of the MCJmolecule, wherein an altered level of the MCJ molecule in the biologicalsample compared to the control level indicates the presence or absenceof the metabolic disease or condition in the subject. In someembodiments, the MCJ molecule is an MCJ polypeptide or a nucleic acidthat encodes an MCJ polypeptide. In certain embodiments, the level ofthe MCJ molecule in the biological sample is decreased compared to thecontrol level. In some embodiments, the level of the MCJ molecule in thebiological sample is increased compared to the control level. In someembodiments, measuring the level of the MCJ molecule includes measuringthe amount and/or function of the MCJ molecule. In some embodiments, themethod also includes selecting a treatment strategy for the subjectbased, at least in part, on the determination of the presence of absenceof the metabolic disease or condition in the subject. In certainembodiments, the treatment strategy includes administering to thesubject a medicament or behavioral treatment for the metabolic diseaseor condition. In some embodiments, the treatment strategy includesceasing administration to the subject of a medicament or behavioraltreatment for the metabolic disease or condition. In some embodiments,the metabolic disease or condition is overweight, weight gain, obesity,non-alcoholic fatty liver disease, diabetes, insulin-resistance,alcoholic fatty liver disease, dyslipidemia, steatosis (e.g., liversteatosis, heart steatosis, kidney steatosis, muscle steatosis),abeta-lipoproteinemia, glycogen storage disease, Weber-Christiandisease, lipodystrophy; a liver disease, liver inflammation, hepatitis,steatohepatitis, Hepatitis C, Genotype 3 Hepatitis C, Alpha1-antitrypsin deficiency, acute fatty liver of pregnancy, Wilsondisease; a kidney disease; a heart disease, hypertension, ischemia,heart failure, cardiomyopathy; poisoning; HIV; a neurodegenerativedisease, Parkinson's disease, Alzheimer's disease; cancer, or physicalexercise. In some embodiments of the invention, the metabolic disease orcondition is high cholesterol. In some embodiments, the metabolicdisease or condition is an eating disorder, anorexia, starvation,malnutrition, total parenteral nutrition, severe weight loss,underweight, re-feeding syndrome; gastrointestinal surgery-mediatedmetabolic alterations, jejuno-ilial bypass, gastric bypass; andinflammatory/infectious conditions, jujunal diverticulosis withbacterial overgrowth, or inflammatory bowel disease. In certainembodiments, the method also includes treating the metabolic disease orcondition the presence of which is determined in the subject.

The present invention is not intended to be limited to a system ormethod that must satisfy one or more of any stated objects or featuresof the invention. It is also important to note that the presentinvention is not limited to the exemplary or primary embodimentsdescribed herein. Modifications and substitutions by one of ordinaryskill in the art are considered to be within the scope of the presentinvention.

BRIEF DESCRIPTION OF THE SEQUENCES

SEQ ID NO:1 is amino acid sequence of human DNAJ domain-containingprotein MCJ set forth as GENBANK® Accession No. AAD38506.1.

SEQ ID NO:2 is mRNA sequence of human DNAJ domain-containing protein MCJset forth as GENBANK® Accession No. AF126743.1.

SEQ ID NO:3 is amino acid sequence of a mouse DNAJ domain-containingprotein.

SEQ ID NO:4 is amino acid sequence of a human DnaJ (Hsp40) homolog ofsubfamily C set forth as GENBANK® Accession No. AAH95400.1.

SEQ ID NO:5 is nucleotide sequence of human DnaJ (HSP40) homolog ofsubfamily C set forth as GENBANK® AccessionNo.BC095400.1.

SEQ ID NO:6 forward primer for region from MCJ intron 1.

SEQ ID NO:7 reverse primer for region from MCJ intron 1.

SEQ ID NO:8 forward primer for MCJ.

SEQ ID NO:9 reverse primer for MCJ.

SEQ ID NO:10 Probe for MCJ.

SEQ ID NO:11: forward primer for MCJ.

SEQ ID NO:12 reverse primer for MCJ.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1A-G shows amino acid sequences, blot images, and a graphillustrating specific tissue distribution of mouse and humanMCJ/DnaJC15. FIG. 1A shows alignment of protein sequences of humanMCJ/DnaJC15 (top sequence) SEQ ID NO:1, and its ortholog in mouse(bottom sequence) SEQ ID NO:3). Differences in the amino acids of thetwo sequences are shown in alignment comparison. FIG. 1B shows Northernblot analysis of mouse normal tissue poly-A mRNA using a specific mousemcj probe. FIG. 1C shows Northern blot analysis of human normal tissuepoly-A mRNA using a specific human mcj probe. FIG. 1D is a graph showingrelative MCJ expression obtained using of real time RT-PCR for mcj usingRNA from mouse B cells, CD4 T cells and CD8 T cells. mRNA levels werenormalized to β-2 microglobulin. FIG. 1E shows blot of whole cellextracts from 293T cells transfected with a mouse mcj expressing plasmid(mMCJ) or an empty plasmid (Cont) that were examined for mouse MCJexpression (MCJ) by Western blot analysis. Actin was examined as aloading control. FIG. 1F shows blot showing endogenous MCJ proteinexpression in mouse liver, heart, kidney and lung that was examined byWestern blot analysis. Actin expression was examined as a loadingcontrol. FIG. 1G is a blot showing endogenous MCJ protein expression inmouse CD4 T cells and CD8 T cells that was examined by Western blotanalysis.

FIG. 2A-I shows photomicrographic images and blots demonstrating thatendogenous MCJ localizes to the mitochondria. FIGS. 2A and B showsresults of immunoelectron microscopy analysis of endogenous MCJ inpurified CD8 T cells (FIG. 2A) and heart (FIG. 2B) from wild type mice.Electron dense gold particle represent MCJ (arrows point to arepresentative immunoreactivity). Small right panels of FIG. 2A and FIG.2B represent a higher magnification of the inset area in the leftpanels. m=mitochondria; mf=myofibrils. 20,000× magnification. Barsindicate 200 nm scale. FIG. 2C shows results of immunoelectronmicroscopy analysis of MCJ in MCJ-transfected 293T cells. Electron densegold particle represents MCJ. Bar indicates 200 nm scale. A swollenmitochondrion (m) is shown. FIG. 2 also shows results of confocalmicroscopy analysis for MCJ (FIG. 2D) and mitotracker (FIG. 2E) inMCJ-transfected 293T cells with FIG. 2F showing overlay of both MCJ andmitotracker. Nuclei were stained with TOPRO (blue). FIG. 2G-I are blotsshowing MCJ expression in purified cytosolic (Cyt) and mitochondrial(Mito) extracts from (FIG. 2G) mouse heart, (FIG. 2H) mouse CD8 T cells,and (FIG. 2I) human MCF7 cells that were examined by Western blotanalysis. GAPDH was used as a marker for the cytosolic fraction andCoxIV as a marker for mitochondrial fraction.

FIG. 3A-J provides graphs showing that MCJ depolarizes mitochondria anddecreases ATP levels. FIG. 3A is a graph showing intracellular ATPlevels in MCF7 cells and MCF7/siMCJ cells (10⁴ cells) incubated inmedium. FIG. 3B is a graph showing intracellular ATP levels inMCF7/siMCJ cells after incubation (4 h) in medium or rotenone (Roten)(10 μM). FIG. 3C is a graph showing intracellular ATP levels in 293Tcells transfected (16 h) with a MCJ-expressing construct (MCJ) or acontrol (Cont). FIG. 3D is a graph showing membrane potential asdetermined by Tetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE)staining and flow cytometry analysis of 293T cells transfected (16 h)with MCJ-expressing (first, shown unshaded) or a control (second, showncrosshatched) constructs. FIG. 3E is a graph showing results of MMP infreshly isolated WT CD8 T cells (filled histogram) and WT CD4 T cells(solid line histogram) that were determined by staining with TMRE andflow cytometry analysis. FIG. 3F is a graph showing results of mROS infreshly isolated WT CD8 T cells (filled histogram) and WT CD4 T cells(solid line histogram) that were determined by staining withMitoSox®-Red and flow cytometry analysis. FIG. 3G is a graph showingresults of MMP in freshly isolated WT CD8 T cells (filled histogram) andMCJ KO CD8 T cells (solid line histogram) which were determined as inFIG. 3E. FIG. 3H is a graph showing results of mROS in freshly isolatedWT CD8 T cells (filled histogram) and MCJ KO CD8 T cells (solid linehistogram) which were determined as in FIG. 3F. FIG. 3I is a graphshowing results of MMP in freshly isolated WT CD4 T cells (filledhistogram) and MCJ KO CD4 T cells (solid line histogram). FIG. 3J is agraph showing results of mROS in freshly isolated WT CD4 T cells (filledhistogram) and MCJ KO CD4 T cells (solid line histogram) was determinedas above. Data are representative of three independent experiments.

FIG. 4A-C provides images of blots showing results of (FIG. 4A) Southernblot analysis showing the MCJ targeted (˜11 Kb) and wild type (˜16 Kb)alleles in wild type (+/+), heterozygous (+/−) and MCJ KO (−/−) mice.FIG. 4 B shows blots from Western blot analysis for MCJ in whole cellextracts from liver, heart, CD4 and CD8 T cells from wild type (WT) andMCJ knockout (KO) mice. Actin was analyzed as loading control. FIG. 4Cshows a blot obtained using RT-PCR for MCJ using RNA from CD8 T cellsisolated from WT, MCJ KO, and MCJ heterozygous mice. Hypoxanthinephosphoribosyltransferase (HPRT) expression was examined as control.

FIG. 5A-B shows plots in FIG. 5A demonstrating CD4 and CD8 T celldistribution in lymph nodes from wild type (WT) and MCJ knock out (KO)mice was analyzed by flow cytometry. Numbers indicate the relativepercentage of cells in each quadrant populations. FIG. 5B is a graphshowing viability of wild type (WT) (thick line) and MCJ knock out (KO)(thin line) CD8 T cells after activation (3 days) with anti-CD3 andanti-CD28 Abs using the UV-Blue dye in flow cytometry analysis.

FIG. 6A-G provides graphs demonstrating that the loss of MCJ maintainshigh mitochondrial metabolism in rested effector CD8 T cells. FIG. 6 Ais a graph showing proliferation of purified CD8 T cells from wild type(WT) and MCJ knock out (KO) mice in response to anti-CD3 and anti-CD28Abs as determined by ³H-Thymidine incorporation. FIG. 6B shows graphs ofMMP in WT CD8 T cells (thin line), MCJ KO CD8 T cells (solid line), andWT CD4 T cells (filled histogram) after activation with anti-CD3 andanti-CD28 Abs for 1 day (left) or 2 days (right), as determined by TMREstaining. FIGS. 6C and D are graphs showing results from WT CD8 T cells(filled histogram) and MCJ KO CD8 T cells (solid line) that wereactivated with anti-CD3 and anti-CD28 Abs for 2 days, washed andincubated in medium alone for 0 h (left panel), 24 h (middle panel) or48 h (right panel). MMP (FIG. 6C) and mROS (FIG. 6D) were examined asdescribed in FIG. 3 . FIG. 6E is a graph showing wild type (WT) and MCJknock out (KO) CD8 T cells that were activated with anti-CD3 andanti-CD28 Abs (2 days), washed, counted and incubated in medium for 12h. Intracellular ATP levels in 10⁴ cells were determined. FIG. 6F is agraph showing results when wild type (WT) and MCJ knock out (KO) CD8 Tcells were activated for 2 days, washed and equal number of cells wasincubated in medium alone for 0, 24 or 48 h. Cell survival wasdetermined by the number of live cells recovered after different periodsof time in medium relative to the number of cells plated at day 2. FIG.6G is a graph of results of CD8 T cells from Thy1.1⁺ OT-1 (WT) and MCJKO Thy1.1/1.2⁺ OT-I (KO) mice were activated with anti-CD3 and anti-CD28Abs for 2 days, expanded in the presence of IL-2 (25 U/ml) for 2 moredays. Equal number of cells were mixed and transferred into Thy1.2⁺ wildtype mice. After 15 days, lymph nodes (LN) and spleen were harvested andthe frequency of donor cells was determined by flow cytometry. Thepercentage of large cells among the corresponding donor cells is shown.Symbols represent individual mice. * denotes p<0.05. Statisticalsignificance between WT and KO lymph nodes (LN) or between WT and KOspleens was determined by student's t test. Data presented here arerepresentative of at least 2 or 3 independent experiments.

FIG. 7 provides graphs showing results from mROS in wild type (WT) CD8 Tcells (thin line), MCJ knock out (KO) CD8 T cells (solid line) and WTCD4 T cells (filled histogram) after activation with anti-CD3 andanti-CD28 Abs for 1 day (left) or 2 days (right), as determined byMitoSox® Red staining.

FIG. 8A-D provides photomicrographic images and graphs demonstratingthat MCJ deficiency enhances liver lipid metabolism during fasting. FIG.8A shows images of liver histology by H&E staining from wild type (WT)and MCJ knock out (KO) mice in normal or fasted (36 h) conditions. Barsindicate 200 μm (normal conditions, 100× magnification), 100 μm (normalconditions, 200× magnification), 100 μm (fasted conditions, 100×magnification) and 50 μm (fasted conditions, 200× magnification). FIG.8B shows micrographic sections from frozen livers of fasted (36 h) wildtype (WT) and MCJ knock out (KO) mice that were stained with Oil Red Ofor detection of lipids. Dark inclusions indicate lipids and higherlevels were seen in the WO than in the KO samples. FIGS. 8C and D aregraphs showing serum triglyceride levels (FIG. 8C) and FFA (FIG. 8D) infasted WT and MCJ KO mice (n=4). * denotes p<0.05. Statisticalsignificance was determined by student's t test. FIG. 9A-B showsphotographic and photomicrographic images of brown fat in normally fedwild type and MCJ knock out (KO) mice. FIG. 9A shows images of brown fat(arrows) in wild type and MCJ KO mice and FIG. 9B shows histology ofbrown fat in wild type and MCJ KO mice under normal feeding conditions.Bars indicate 100 μm scale.

FIG. 10A-B shows photographic and photomicrographic images of brown fatin fasted wild type and MCJ knock out (KO) mice fasted for 36 hours.FIG. 10A shows images of brown fat (arrow) in wild type and MCJ KO miceand FIG. 10B shows histology of brown fat in wild type and MCJ KO mice.Bars indicate 100 μm scale.

FIG. 11A-K shows graphs and photomicrographic sections indicating thatMCJ deficiency promotes glyconeogenesis during fasting. FIG. 11A showspercentages of total body weight loss after 36 h of fasting relative tothe initial weight in wild-type (WT) and MCJ knock-out (KO) mice (n=3).FIG. 11B shows total body weight in WT and MCJ KO mice prior to fasting.FIG. 11C shows comparison of glucose levels in WT and MCJ KO mouse blood12 h after fasting. FIG. 11D shows comparison of WT and MCJ KO glucoselevels in blood prior to and during fasting. FIG. 11E shows comparisonof ATP concentrations in liver extracts from WT and MCJ KO mice afterfasting (36 h). FIG. 11F shows PAS staining in liver sections from WTand MCJ KO mice after fasting. FIG. 11G shows comparison of glycogencontents in liver extracts from WT and MCJ KO mice (n=3) after fasting(36 h) or normal feeding. FIG. 11H shows percentages of liver weightversus total body weight in WT and MCJ KO mice after fasting (36 h) ornormal feeding. FIG. 11I shows Western blot analysis for glycogensynthase (GS) and PEPCK in livers from WT and MCJ KO mice normally fed(Cont) or after fasting (Fast) for 36 h. Livers from two mice are shownfor the fasting condition. FIG. 11J shows cholesterol content in liversof WT and MCJ KO mice (n=5) after 4 weeks on a high-cholesterol diet.FIG. 11K shows cholesterol contents in livers of WT and MCJ KO mice(n=4) fed a normal diet. *, P<0.05. Statistical significance wasdetermined by the Student t test. The error bars indicate standarddeviations.

FIG. 12A-F provides blots, photomicrographic images, and graphsdemonstrating delayed mammary tumor growth in MCJ deficient mice. FIG.12A is a blot showing MCJ expression in normal mammary gland (N) from awild type (WT) female mouse, or in mammary tumors isolated from twoMMTV-PyMT mice (T1 and T2) that were examined by Western blot analysisusing whole cell extracts. FIG. 12B is a blot showing results of MCJexpression examined by Western blot analysis using whole cell extracts(WE), cytosolic extracts (Cyt), and mitochondria extracts (Mito) fromMMTV-PyMT tumors. CoxIV and GAPDH were also examined. FIG. 12C is a blotshowing results of MCJ expression in mitochondrial extracts from normalmammary tissues (N) or from a MMTV-PyMT tumor (T) examined by Westernblot analysis using whole cell extracts. FIG. 12D is a Kaplan-Meiersurvival curve of MMTV-PyMT wild type (WT) and MCJ KO MMTV-PyMT [MCJknock out (KO)] mice (n=8). Statistical significance was determined bylogrank test. FIG. 12E provides photomicrographic images showinghistology (H&E) of mammary tumors from MMTV-PyMT and MCJ KO MMTVPyMTmice. FIG. 12F is a graph showing a ratio of ATP levels in mitochondriaextracts relative to ATP levels in cytosolic extracts from tumors ofMMTV-PyMT wild type (WT) mice (n=5) and MCJ MMTV-PyMT knock out (KO)mice (n=6). * denotes p<0.05. Statistical significance was determined bystudent's t test.

FIG. 13A-G provides blots and graphs of results demonstrating that MCJis a repressor of the mitochondria respiratory chain Complex I activity.FIG. 13A is a blot showing the presence of MCJ, NDUFA9 and NDUFS3 inimmunoprecipitates of Complex I from mitochondrial extracts generatedfrom WT and MCJ KO hearts was examined by Western blot analysis. FIG.13B is a graph showing relative Complex I activity in mitochondrialextracts (10 μg) from wild type (WT) and MCJ knock out (KO) mousehearts. FIG. 13C is a blot showing expression of NDUFA9 and MCJ in heartmitochondrial extracts from wild type (WT) and MCJ knock out (KO) micethat were examined by Western blot analysis. FIG. 13D is a graph showingrelative Complex I activity in mitochondrial extracts (5 μg) fromfreshly isolated wild type (WT) CD4, wild type (WT) CD8 and MCJ knockout (KO) CD8 T cells. FIG. 13E is a blot showing expression of MCJ,NDUFA9, NDUFS3 and actin in mitochondrial extracts from wild type (WT)CD4, wild type (WT) CD8, and MCJ knock out (KO) CD8 T cells that wereexamined by Western blot analysis. FIG. 13F is a blot showing expressionof NDUFA9 and MCJ in mitochondrial extracts from MCF7 cells andMCF7/siMCJ cells (siMCJ) that were examined by Western blot analysis.FIG. 13G is a graph showing relative Complex I activity in mitochondrialextracts purified from MCF7 and MCF7/siMCJ cells.

DETAILED DESCRIPTION

It has now been discovered that methods and compounds that modulate MCJpolypeptide activity are useful to treat diseases and conditionscharacterized by metabolic dysregulation. MCJ/DnaJC15 functions as aunique mitochondrial co-chaperone that negatively regulates Complex I ofthe mitochondrial respiratory chain and ATP production. It has also beendetermined that this negative feedback attenuates mitochondrialrespiration as a response to selective metabolic pressures. Thus, MCJdeficiency impedes slowing of metabolism in response to fasting and anMCJ deficiency enhances mitochondrial lipid oxidation, which results indecreased lipid accumulation in the liver and protection from steatosis.Increased mitochondrial respiration caused by MCJ deficiency has alsobeen shown to delay mammary tumor growth.

The DnaJ family of co-chaperones is the largest with more than 49members identified in human. Although the DnaJ domain is highlyconserved in evolution, the members of this family vary in thenon-conserved domains, their tissue expression and cellularlocalization. The function of most vertebrate DnaJ members in primarytissues remains to be elucidated. In this study the murine ortholog forhuman MCJ (DnaJC15) and its specific tissue distribution that isconserved between the two species are now described for the first time.It has now been demonstrated that MCJ provides a negative feedback tothe mitochondrial respiratory chain and ATP production by interferingwith Complex I activity. More importantly, the results show that MCJ isa key regulator of metabolic switches in vivo, a novel function that hasnot been previously reported for any other member of the DnaJ family.Cellular localization influences the specific functions of DnaJ familymembers. Using immuno-EM and biochemical analysis in a variety ofprimary tissues and cells, it has now been shown that MCJ localizes tothe mitochondria, in proximity to the inner membrane.

Methylation-Controlled J protein (MCJ)/DnaJC15 is a member of the DnaJCsubfamily of cochaperones. MCJ is a small protein of 150 amino acids anda unique member of the DnaJC family. It contains a J-domain located atthe C-terminus, as opposed to the common N-terminal position, and itsN-terminal region has no homology with any other known protein. Inaddition, MCJ also contains a transmembrane domain while most DnaJproteins are soluble. Phylogenetic studies have shown that MCJ is onlypresent in vertebrates where it is highly conserved (Hatle et al.,2007). The amino acid sequence of human DNAJ domain-containing proteinMCJ of GENBANK® Accession No. AAD38506.1 is set forth herein as SEQ IDNO:1. SEQ ID NO:2 is mRNA sequence of human DNAJ domain-containingprotein MCJ set forth as GENBANK® Accession No. AF126743.1.

GENBANK® Accession No. AAH95400.1 provides amino acid sequence of ahuman DnaJ (Hsp40) homolog of subfamily C, which is provided herein asSEQ ID NO:4. SEQ ID NO:5 is nucleotide sequence of human DnaJ (HSP40)homolog of subfamily C set forth as GENBANK® AccessionNo. BC095400.1.

MCJ/DnaJC15 is the first DnaJ transmembrane member shown to reside inmitochondria both in human and mouse. Furthermore, it has now been shownthat one of the functions of MCJ is to provide a negative feedback toComplex I in order to attenuate its activity when needed. Thus, in theabsence of MCJ there is a significant increase in Complex I activity. Anumber of associated proteins have been identified as necessary forComplex I to be fully active (McKenzie and Ryan, 2010 IUBMB Life 62,497-502), MCJ is one of a few molecules that repress its activity.Intriguingly, a significant change of Complex I during evolution was theacquisition of both inactive and active forms in vertebrates, while onlythe active form has been identified in prokaryotes (Clason et al., 2009J Struct Biol 169, 81-88; Ohnishi et al., 1998 Biochim Biophys Acta1365, 301-308). Thus, mammalian Complex I is a mixture of both activeand inactive forms. The mechanisms that regulate the balance betweenboth forms remain unclear. Because MCJ originated in vertebrates andrepresses Complex I activity, it is hypothesized that MCJ regulates thebalance between Complex I active and inactive forms.

Among other findings, it has now been shown that (1) MCJ/DnaJC15 is anovel endogenous negative regulator of mitochondrial Complex I; (2) MCJdeficiency results in enhanced mitochondrial respiration and ATPproduction; (3) MCJ deficiency accelerates lipid metabolism in the liverand prevents the development of steatosis; and (4) negative regulationof mitochondrial oxidative phosphorylation by MCJ affects cancerprogression.

The invention pertains, in part, to modulating activity (e.g., levelsand/or function) of MCJ/DnaJC15 (also referred to herein as MCJpolypeptide) to regulate metabolic activity in cells, tissues, organs,and subjects. Compositions, compounds, and methods of the invention maybe used for treating a subject having, or at risk of having, a metabolicdisease or condition that may be characterized by abnormal levels of MCJactivity or undesirable levels of MCJ polypeptide activity. Theinvention, in part, also relates to methods of diagnosing and assessingthe status of metabolic diseases and conditions that may becharacterized by abnormal MCJ polypeptide activity. Thus, methods andcompounds of the invention are useful to treat and assess such metabolicdiseases and conditions in subjects. The invention in part, also relatesto modulating MCJ polypeptide activity from an initial activity level ina subject to an activity level that is effective to reduce or eliminatesymptoms of a metabolic disease or condition and/or to prevent onset ofsymptoms of a metabolic disease or condition.

As used herein, a subject shall mean a human or vertebrate mammalincluding but not limited to a dog, cat, horse, goat, and primate, e.g.,monkey. Thus, the invention can be used to treat diseases or conditionsin human and non-human subjects. For instance, methods and compositionsof the invention can be used in veterinary applications as well as inhuman prevention and treatment regimens. In some embodiments of theinvention, the subject is a human. In some embodiments of the invention,a subject does not have cancer. In certain embodiments of the invention,a subject has cancer. In certain embodiments, a subject is undergoingcaloric reduction, e.g., fasting. As used herein, the term “fasting”means a reduced caloric intake, which may include a reduction of up to5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, or more of the normalcaloric intake for a given subject over a predetermined period of time.In some embodiments of the invention a predetermined period of time maybe 1, 2, 3, 4, 5, 6, 7 or more days; 1, 2, 3, 4 or more weeks; 1, 2, 3,4, 5, 6, 7, 8, 9, 10, 11, 12, or more months; and/or 1, 2, 3, 4, 5, ormore years. A skilled artisan is able to assess the level of normalcaloric intake and a level of reduced caloric intake for a given subjectusing art-known standards and recommendations, e.g., height/weighttables, etc. As used herein, fasting may be considered a behavioraltreatment for a metabolic disease or condition characterized by abnormalMCJ polypeptide activity.

Non-limiting examples of subjects to which the present invention can beapplied are subjects who are diagnosed with, suspected of having, or atrisk of having, a metabolic disease or condition. Methods of theinvention may be applied to a subject who, at the time of treatment, hasbeen diagnosed as having a metabolic disease or condition, or a subjectwho is considered to be at risk for having or developing a metabolicdisease or condition.

In some aspects of the invention, a subject having a metabolic diseaseor condition is a subject that has detectable abnormality in metabolismas compared to a normal control. In certain aspects of the invention, asubject may have no significant difference compared to a normal control,but it is desired to alter the subject's metabolic activity usingmethods provided herein. For example, a subject may have a metabolicdisease such as diabetes or fatty liver disease that is associated withan abnormality in the subject's metabolic activity compared to a normalcontrol that does not have diabetes or fatty liver disease. In anotherexample, a subject may have a metabolic condition, such as increasedexercise, for which it is desirable to increase metabolism in thesubject.

In some aspects of the invention, a subject is at risk of having ordeveloping a metabolic disease or condition. A subject at risk ofdeveloping a metabolic disease or condition is one who has an increasedprobability of developing the disease or condition, compared to acontrol risk of developing the metabolic disease or condition. In someembodiments of the invention, a level of risk may be statisticallysignificant compared to a control level of risk. A subject at risk mayinclude, for instance, subjects having a genetic abnormality, thepresence of which has been demonstrated to have a correlative relationto a higher likelihood of developing a metabolic disease or condition;subjects having a family and/or personal medical history of themetabolic disease or condition; subjects exposed to agents such aschemical toxins, or activities; and/or subjects who have previously beentreated for the metabolic disease or condition and are in apparentremission.

In some metabolic diseases or conditions MCJ polypeptide activity in acell, tissue, or subject may be higher relative to MCJ polypeptideactivity in a cell, tissue, or subject that does not have the metabolicdisease or condition. Thus, in some diseases and conditions that can betreated with methods and compounds of the invention, a higher thannormal MCJ polypeptide activity may be characteristic for the metabolicdisease or condition, and may also be diagnostic for the metabolicdisease or condition. Certain metabolic diseases or conditions may notbe characterized by higher than normal MCJ activity level in a cell,tissue, or subject, but can be treated by decreasing MCJ polypeptideactivity in a given subject. For example, a subject may have normallevels of MCJ polypeptide activity compared to a control level from asample, tissue, or subject that does not have the metabolic disease orcondition, but decreasing the level in the subject, may treat themetabolic disease or condition in that subject.

Thus, some embodiments of the invention include methods of administeringa compound to the cell, tissue or subject in an amount effective todecrease MCJ polypeptide activity in the cell, tissue, or subject as atreatment for the disease or condition. Metabolic diseases andconditions such as overweight, weight gain, obesity, non-alcoholic fattyliver disease, diabetes, insulin-resistance, alcoholic fatty liverdisease, dyslipidemia, steatosis (e.g., liver steatosis, heartsteatosis, kidney steatosis, muscle steatosis), abeta-lipoproteinemia,glycogen storage disease, Weber-Christian disease, lipodystrophy; liverdiseases including but not limited to: liver inflammation, hepatitis,steatohepatitis, Hepatitis C, Genotype 3 Hepatitis C, Alpha1-antitrypsin deficiency, acute fatty liver of pregnancy, and Wilsondisease; kidney diseases; heart diseases: including but not limited tohypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV;neurodegenerative diseases: including but not limited to Parkinson'sdisease, Alzheimer's disease; physical exercise; and cancer may betreated by decreasing MCJ-polypeptide activity in a cell, tissue, orsubject.

In some metabolic diseases or conditions MCJ polypeptide activity in acell, tissue, or subject may be lower relative to MCJ polypeptideactivity in a cell, tissue, or subject that does not have the metabolicdisease or condition. Thus, in some diseases and conditions that can betreated with methods and compounds of the invention, a lower than normalMCJ polypeptide activity may be characteristic for the metabolic diseaseor condition, and may also be diagnostic for the metabolic disease orcondition. Certain metabolic diseases or conditions may not becharacterized by lower than normal MCJ activity level in a cell, tissue,or subject, but can be treated by increasing MCJ polypeptide activity ina given subject. For example, a subject may have normal levels of MCJpolypeptide activity compared to a control level from a sample, tissue,or subject that does not have the metabolic disease or condition, butincreasing the level in the subject, may treat the metabolic disease orcondition in that subject.

Thus, some embodiments of the invention include methods of administeringa compound to the cell, tissue or subject in an amount effective toincrease MCJ polypeptide activity in the cell, tissue, or subject as atreatment for the disease or condition. Metabolic diseases andconditions such as eating disorders: including but not limited toanorexia, starvation, malnutrition, total parenteral nutrition, severeweight loss, underweight, re-feeding syndrome; gastrointestinalsurgery-mediated metabolic alterations: including but not limited to:jejuno-ilial bypass, gastric bypass; and inflammatory/infectiousconditions: including but not limited to jujunal diverticulosis withbacterial overgrowth, and inflammatory bowel disease may be treated byincreasing MCJ-polypeptide activity in a cell, tissue, or subject.

MCJ polypeptide activity (e.g., level of MCJ polypeptide and/or functionof MCJ polypeptide) can be determined and compared to control values ofMCJ polypeptide activity according to the invention. A control may be apredetermined value, which can take a variety of forms. It can be asingle cut-off value, such as a median or mean. It can be establishedbased upon comparative groups, such as in groups having normal amountsof MCJ polypeptide activity and groups having abnormal amounts of MCJpolypeptide activity. Another example of comparative groups may begroups having one or more symptoms of or a diagnosis of a metabolicdisease or condition and groups without having one or more symptoms ofor a diagnosis of a metabolic disease or condition. Another comparativegroup may be a group with a family history of a metabolic disease orcondition and a group without such a family history. A predeterminedvalue can be arranged, for example, where a tested population is dividedequally (or unequally) into groups, such as a low-risk group, amedium-risk group and a high-risk group or into quadrants or quintiles,the lowest quadrant or quintile being individuals with the lowest risk(e.g. of a metabolic disease or condition) and the lowest amounts of MCJpolypeptide activity and the highest quadrant or quintile beingindividuals with the highest risk (e.g. of a metabolic disease orcondition) and highest amounts of MCJ polypeptide activity.

The predetermined value, of course, will depend upon the particularpopulation selected. For example, an apparently healthy population willhave a different ‘normal’ range than will a population that is known tohave a condition related to abnormal MCJ protein expression or presence.Accordingly, the predetermined value selected may take into account thecategory in which an individual or cell falls. Appropriate ranges andcategories can be selected with no more than routine experimentation bythose of ordinary skill in the art. As used herein, “abnormal” meanssignificantly different as compared to a normal control. By abnormallyhigh MCJ polypeptide activity it is meant high relative to a selectedcontrol, and may include an increase in activity of at least 0.1%, 0.5%,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, 150%, 200%,or more, in a subject or cell as compared to the level in a normalcontrol. By abnormally low MCJ polypeptide activity it is meant lowrelative to a selected normal control, and include a decrease of atleast 0.5%, 1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100% ina subject or cell as compared to the level in a normal control.Typically, the control will be based on apparently healthy normalindividuals in an appropriate age bracket or apparently healthy cells.

In some aspects of the invention, values of MCJ polypeptide activitydetermined for a subject may serve as control values for laterdeterminations of MCJ polypeptide activity in that same subject, thuspermitting assessment of changes from a “baseline” MCJ polypeptideactivity in a subject. Thus, an initial MCJ polypeptide activity levelmay be determined in a subject and methods and compounds of theinvention may be used to decrease the level of MCJ polypeptide activityin the subject, with the initial level serving as a control level forthat subject. Using methods and compounds of the invention, the MCJpolypeptide activity in the subject may be decreased by at least 0.5%,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, or more compared to theinitial level as a treatment for a metabolic disease or condition in thesubject. Similarly, an initial MCJ polypeptide activity level may bedetermined in a subject and methods and compounds of the invention maybe used to increase the level of MCJ polypeptide activity in thesubject, with the initial level serving as a control level for thatsubject. Using methods and compounds of the invention, the MCJpolypeptide activity in the subject may be increased by at least 0.5%,1%, 5%, 10%, 20%, 30%, 40%, 50%, 60%, 70%, 80%, 90%, 100%, or morecompared to the initial level as a treatment for a metabolic disease orcondition in the subject.

It will be understood that controls according to the invention may be,in addition to predetermined values, samples of materials tested inparallel with the experimental materials. Examples include samples fromcontrol populations or control samples generated through manufacture tobe tested in parallel with the experimental samples.

It now has been determined that the level of MCJ polypeptide activitymitochondria of cells correlates with the presence or absence of ametabolic disease or condition, and that an increased amount of MCJpolypeptide activity corresponds to an increase in risk of a metabolicdisease or condition in subjects. A lower level of MCJ polypeptideactivity in mitochondria of cells in a subject has also now beencorrelated with a more positive prognosis for a subject than theprognosis if the subject's cells have a higher level of MCJ polypeptideactivity. For example, although not intended to be limiting, a lowerlevel of MCJ polypeptide activity increases the rate of lipid metabolismin the liver and prevents development of steatosis, which leads to abetter prognosis for the subject than if a higher level of MCJpolypeptide activity. Thus, a higher level of expression and function ofMCJ in cells is associated with a higher occurrence of metabolic diseaseand abnormality and a worse prognosis in the subject. One of ordinaryskill in the art will recognize that the terms higher, lower, reduced,increased, may be represent relative levels or values as compared tocontrol levels or values.

Although not intending to be bound by any particular theory, it isbelieved that MCJ resides in the mitochondria where it serves as anegative regulator of Complex I of the mitochondrial respiratory chain.Loss of MCJ leads to increased Complex I activity, hyperpolarization ofmitochondria and increased generation of ATP. Enhanced mitochondrialoxidative phosphorylation as a source of energy in the absence of MCJaccelerates fatty acid oxidation and prevents accumulation of lipids inthe liver during starvation. In addition, MCJ deficiency has also beenfound to delay mammary tumor growth correlating with the inability toattenuate mitochondrial function.

In addition, although not intending to be bound by any particulartheory, it is believed that modulating MCJ polypeptide activity is alsocorrelated with modulating cytotoxic T (CD8⁺ T) cell function in cells,tissues and subjects. In some embodiments, methods of the invention mayinclude administering to a subject in need of such treatment anMCJ-modulating compound in an amount effective to increase or decreasean activity of an MCJ polypeptide in a CD8⁺ T cell. An increase in MCJpolypeptide activity has been found to increase depolarization of amitochondrion in the CD8⁺ T cell and a decrease in MCJ polypeptideactivity has been found to decrease depolarization of a mitochondrion inthe CD8⁺ T cell. Thus, administering a compound that modulates MCJpolypeptide activity can be used to increase or decrease depolarizationof the mitochondrion and thus to modulate cytotoxic T cell function inthe subject.

Treatment

The invention in some aspects relates to methods for modulating MCJpolypeptide activity in a cell, tissue, and/or subject. As used hereinthe term “modulating” means changing a level of an MCJ polypeptideactivity (e.g., MCJ polypeptide level and/or function) in a cell. Insome embodiments of the invention, changing MCJ polypeptide activityincludes changing a level of an MCJ polypeptide in a cell or tissue.Thus, increasing activity of MCJ polypeptide in a cell may includeincreasing the level (e.g., amount) of the MCJ polypeptide in the cell.Similarly, decreasing the activity of the MCJ polypeptide in a cell mayinclude decreasing the level (e.g., amount) of the MCJ polypeptide inthe cell. In some embodiments of the invention, a level of MCJpolypeptide can be changed or modulated by increasing expression of anMCJ polypeptide. Thus, some embodiments of the invention methods mayinclude increasing or decreasing the level of an MCJpolypeptide-encoding nucleic acid in a cell, tissue, or subject, whichmay result in an increased activity of MCJ polypeptide in the cell,tissue, or subject. Certain embodiments of the invention methods mayinclude directly increasing or decreasing the level of an MCJpolypeptide in a cell, tissue, or subject, for example, by deliveringthe MCJ polypeptide into the cell, tissue or subject, to treat ametabolic disease or condition characterized by abnormal MCJ polypeptideactivity. As set forth elsewhere herein, in some embodiments of theinvention, cells that may be treated with a method of the invention toincrease or decrease a level of an MCJ polypeptide or its activity mayinclude, but are not limited to, liver cells, muscle cells, cardiaccells, circulatory cells, neuronal cells, glial cells, fat cells, skincells, hematopoietic cells, epithelial cells, sperm, oocytes, musclecells, adipocytes, kidney cells, hepatocytes, or pancreas cells. In someembodiments of the invention, a cell is a cancer cell, an example ofwhich may be a tumor cell.

As used herein, the terms “treat”, “treated”, or “treating” when usedwith respect to a disorder such as a metabolic disease or condition thatmay be characterized by abnormal MCJ polypeptide activity may refer to aprophylactic treatment that decreases the likelihood of a subjectdeveloping the disease or condition, and also may refer to a treatmentafter the subject has developed the disease or condition in order toeliminate or reduce the level of the disease or condition, prevent thedisease or condition from becoming more advanced (e.g., more severe),and/or slow the progression of the disease compared to in the absence ofthe therapy.

In certain embodiments of the invention, changing MCJ polypeptideactivity includes increasing or decreasing functioning of an MCJpolypeptide in a cell, tissue, or subject. In some such embodiments, thelevel of the MCJ polypeptide does not change, but the function of one ormore of the MCJ polypeptides in a cell may be altered, for exampleeither increased or decreased. Examples of methods that may alter thefunction of an MCJ polypeptide may include, but are not limited tocontacting the MCJ polypeptide with an antibody or functional fragmentthereof that binds to an MCJ polypeptide and alters its function. Forexample, in some embodiments of the invention an antibody that inhibitsMCJ function may be delivered to a cell as part of a treatment regimen.Similarly, in some embodiments of the invention, compounds that enhanceor inhibit MCJ function may be administered to a cell or subject andresult in a modulation of MCJ polypeptide activity. Compounds thatenhance or inhibit an MCJ polypeptide function and/or enhanced orinhibit an MCJ polypeptide level may be referred to herein asMCJ-modulating compounds.

In some embodiments of the invention, an MCJ-modulating compound mayinclude an MCJ molecule, an anti-MCJ polypeptide antibody or functionalfragment thereof, a small molecule MCJ inhibitor, or a small moleculeMCJ enhancer. Examples of MCJ molecules include MCJ polypeptides ornucleic acids that encode MCJ polypeptides. As used herein anMCJ-modulating compound may be a compound that modulates (e.g, increasesor decreases) MCJ polypeptide activity in a cell, tissue, and/orsubject. An MCJ-modulating compound that decreases or reduces MCJpolypeptide activity may be referred to as an MCJ inhibitor compound andan MCJ-modulating compound that increases or enhances MCJ polypeptideactivity may be referred to as an MCJ enhancer compound.

Thus, compounds that increase or decrease an MCJ polypeptide activitymay be administered in an effective amount to a subject in need oftreatment of a metabolic disease or condition. Administering a compoundthat increases or a compound that decreases MCJ polypeptide activity toa subject may reduce a metabolic disease or condition in the subject. Incertain metabolic disease and/or conditions in which it is desirable toreduce MCJ polypeptide activity using a treatment and/or compound of theinvention, an MCJ inhibiting compound may be administered. Non-limitingexamples of such metabolic diseases or conditions include but are notlimited to: overweight, weight gain, obesity, non-alcoholic fatty liverdisease, diabetes, insulin-resistance, alcoholic fatty liver disease,dyslipidemia, steatosis (e.g., liver steatosis, heart steatosis, kidneysteatosis, muscle steatosis), abeta-lipoproteinemia, glycogen storagedisease, Weber-Christian disease, lipodystrophy; liver diseasesincluding but not limited to: liver inflammation, hepatitis,steatohepatitis, Hepatitis C, Genotype 3 Hepatitis C, Alpha1-antitrypsin deficiency, acute fatty liver of pregnancy, and Wilsondisease; kidney diseases; heart diseases: including but not limited tohypertension, ischemia, heart failure, cardiomyopathy; poisoning; HIV;neurodegenerative diseases: including but not limited to Parkinson'sdisease, Alzheimer's disease; physical exercise; and cancer. In someembodiments of the invention, the metabolic disease or condition is highcholesterol and methods of the invention to reduce MCJ activity may beused to reduce cholesterol in a cell, tissue, or subject.

In certain metabolic conditions in which it is desirable to increase MCJpolypeptide activity using a treatment and/or compound of the invention,an MCJ enhancing compound may be administered. Non-limiting examples ofsuch metabolic diseases or conditions include but are not limited to:eating disorders: including but not limited to anorexia, starvation,malnutrition, total parenteral nutrition, severe weight loss,underweight, re-feeding syndrome; gastrointestinal surgery-mediatedmetabolic alterations: including but not limited to: jejuno-ilialbypass, gastric bypass; and inflammatory/infectious conditions:including but not limited to jujunal diverticulosis with bacterialovergrowth, and inflammatory bowel disease.

A compound useful to treat a metabolic disease or condition for which itis desirable to alter or modulate MCJ polypeptide activity may, in someembodiments of the invention be an MCJ polypeptide or nucleic acid thatencodes an MCJ polypeptide. Thus, a method of the invention may includeadministering an exogenous MCJ polypeptide or exogenous MCJpolypeptide-encoding nucleic acid to a subject.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably and thus the term polypeptide may be used to refer to afull-length protein and may also be used to refer to a fragment of afull-length protein. As used herein with respect to polypeptides,proteins, or fragments thereof, and nucleic acids that encode suchpolypeptides the term “exogenous” means the compound is administered toa cell or subject and was not naturally present in the cell or subject.It will be understood that an exogenous MCJ polypeptide or MCJpolypeptide-encoding nucleic acid may be identical to an endogenous MCJpolypeptide or MCJ polypeptide-encoding nucleic acid, respectively, interms of its sequence, but was administered to the cell or subject.

According to some aspects of the invention, full-length MCJ polypeptidesor fragments of full-length MCJ polypeptide may be administered inmethods of the invention. Fragments of the invention may be fragmentsthat retain a distinct functional capability of the polypeptide.Functional capabilities that can be retained in a fragment includeinteraction with antibodies, and interaction with other polypeptides orfragments thereof. Polypeptide fragments may be natural fragments or maybe synthesized using art-known methods, and tested for function usingthe methods exemplified herein. Full-length MCJ and fragments of MCJthat are useful in methods and compositions of the invention may berecombinant polypeptides.

A fragment of a full-length MCJ polypeptide may comprise at least up ton−1 contiguous amino acids of the full-length MCJ polypeptide having aconsecutive sequence found in a wild-type MCJ polypeptide or in amodified MCJ polypeptide sequence as described herein (with “n” equal tothe number of amino acids in the full-length MCJ polypeptide). Thus, forexample, a fragment of a 150 amino acid-long MCJ polypeptide would be atleast 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25,30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 110, 120,130, 140, or 149 (including each integer in between) contiguous aminoacids of the 150 amino acid MCJ polypeptide. In some embodiments, afragment includes the C-terminal region of an MCJ polypeptide. Such MCJpolypeptides that are fragments of full-length MCJ polypeptide may beuseful for a variety of purposes, including for administration asMCJ-modulating compounds and for preparing MCJ-modulating compounds suchas antibodies that bind specifically to synthetic and natural MCJpolypeptides.

A “modified” wild-type or mutant full-length MCJ polypeptide orpolypeptide that is a fragment thereof may include deletions, pointmutations, truncations, amino acid substitutions and/or additions ofamino acids or non-amino acid moieties. Modifications of a polypeptideof the invention may be made by modification of the nucleic acid thatencodes the polypeptide or alternatively, modifications may be madedirectly to the polypeptide, such as by cleavage, addition of a linkermolecule, addition of a detectable moiety, such as a fluorescent label,and the like. Modifications also embrace fusion proteins comprising allor part of the polypeptide's amino acid sequence.

In general, modified polypeptides (e.g. modified MCJ wild-type or mutantpolypeptides) may include polypeptides that are modified specifically toalter a feature of the polypeptide unrelated to its physiologicalactivity. For example, cysteine residues can be substituted or deletedto prevent unwanted disulfide linkages. A residue may be added at the Nor C-terminal end of the polypeptide, for example, a cysteine (C) orother amino acid residue may be added at the extreme C-terminal end of aMCJ polypeptide. MCJ polypeptides can be synthesized with modificationsand/or modifications can be made in an MCJ polypeptide by selecting andintroducing an amino acid substitution, deletion, or addition. Modifiedpolypeptides then can be tested for one or more activities (e.g.,modulating MCJ-polypeptide activity in a cell or subject, treatment of ametabolic disease or condition, altering a mitochondrial membranedepolarization, etc.) to determine which modification provides amodified polypeptide with the desired properties.

The skilled artisan will also realize that conservative amino acidsubstitutions may be made in a polypeptide to provide functionallyequivalent polypeptides, i.e., a modified MCJ polypeptide that retains afunctional capability of an un-modified MCJ polypeptide in a treatmentmethod of the invention. As used herein, a “conservative amino acidsubstitution” refers to an amino acid substitution that does not alterthe relative charge or size characteristics of the polypeptide in whichthe amino acid substitution is made. Modified MCJ polypeptides can beprepared according to methods for altering polypeptide sequence andknown to one of ordinary skill in the art such. Exemplary functionallyequivalent MCJ polypeptides include conservative amino acidsubstitutions of an MCJ polypeptide, or fragments thereof, such as amodified MCJ polypeptide. Conservative substitutions of amino acidsinclude substitutions made amongst amino acids within the followinggroups: (a) M, I, L, V; (b) F, Y, W; (c) K, R, H; (d) A, G; (e) S, T;(f) Q, N; and (g) E, D.

Conservative amino-acid substitutions in an MCJ polypeptide typicallyare made by alteration of a nucleic acid encoding the polypeptide. Suchsubstitutions can be made by a variety of methods known to one ofordinary skill in the art. For example, amino acid substitutions may bemade by PCR-directed mutation, site-directed mutagenesis, or by chemicalsynthesis of a gene encoding the MCJ polypeptide. Where amino acidsubstitutions are made to a small fragment of a polypeptide, thesubstitutions can be made by directly synthesizing the polypeptide. Theactivity of functionally equivalent fragments of MCJ polypeptides can betested by cloning the gene encoding the altered polypeptide into abacterial or mammalian expression vector, introducing the vector into anappropriate host cell, expressing the altered polypeptide, and testingfor a functional capability of the polypeptide as disclosed herein.

In some embodiments of the invention, a level or function of a MCJpolypeptide may be modulated by genetically introducing an MCJpolypeptide into a cell and/or mitochondria, and reagents and methodsare provided for genetically targeted expression of MCJ polypeptides.Genetic targeting can be used to deliver MCJ polypeptides to specificcell types, to specific cell subtypes, to specific spatial regionswithin an organism, and to sub-cellular regions within a cell. Genetictargeting also relates to the control of the amount of an MCJpolypeptide expressed, and the timing of the expression. Someembodiments of the invention include a reagent for genetically targetedexpression of an MCJ polypeptide, wherein the reagent comprises a vectorthat contains a nucleic acid that encodes an MCJ polypeptide or encodesa functional fragment of an MCJ polypeptide.

As used herein, the term “vector” refers to a nucleic acid moleculecapable of transporting between different genetic environments anothernucleic acid to which it has been operatively linked. The term “vector”also refers to a virus or organism that is capable of transporting thenucleic acid molecule. One type of vector is an episome, i.e., a nucleicacid molecule capable of extra-chromosomal replication. Some usefulvectors are those capable of autonomous replication and/or expression ofnucleic acids to which they are linked. Vectors capable of directing theexpression of genes to which they are operatively linked are referred toherein as “expression vectors”. Other useful vectors, include, but arenot limited to viruses such as lentiviruses, retroviruses, adenoviruses,and phages. Vectors useful in some methods of the invention cangenetically insert MCJ polypeptides into dividing and non-dividing cellsand can insert MCJ polypeptides to cells that are in vivo, in vitro, orex vivo cells.

Vectors useful in methods of the invention may include additionalsequences including, but not limited to one or more signal sequencesand/or promoter sequences, or a combination thereof. Expression vectorsand methods of their use are well known in the art. In certainembodiments of the invention, a vector may be a lentivirus comprising anucleic acid or gene that encodes an MCJ polypeptide of the invention ora variant thereof. A lentivirus is a non-limiting example of a vectorthat may be used to create stable cell line. The term “cell line” asused herein is an established cell culture that will continue toproliferate given the appropriate medium.

Promoters that may be used in methods and vectors of the inventioninclude, but are not limited to, cell-specific promoters or generalpromoters. Methods for selecting and using cell-specific promoters andgeneral promoters are well known in the art. A non-limiting example of ageneral purpose promoter that allows expression of an MCJ polypeptide ina wide variety of cell types—thus a promoter for a gene that is widelyexpressed in a variety of cell types, for example a “housekeeping gene”can be used to express an MCJ polypeptide in a variety of cell types.Non-limiting examples of general promoters are provided elsewhere hereinand suitable alternative promoters are well known in the art. In certainembodiments of the invention, a promoter may be an inducible promoter,examples of which include, but are not limited to tetracycline-on ortetracycline-off, etc.

Certain aspects of the invention include methods of administeringantibodies or antigen-binding fragments thereof which specifically bindto an MCJ polypeptide to alter MCJ polypeptide activity, e.g., todecrease MCJ polypeptide activity. In some embodiments of the inventionsuch antibodies or antigen-binding fragments thereof may be administeredto a cell and/or subject to inhibit MCJ polypeptide activity in the celland/or subject. The term “antigen-binding fragment” of an antibody asused herein, refers to one or more portions of an antibody that retainthe ability to specifically bind to an antigen (e.g., an MCJpolypeptide). One may prepare and test an antigen-binding fragment of anMCJ-modulating antibody for use in methods of the invention usingart-known methods and routine procedures. In some embodiments of theinvention, the antibodies are recombinant antibodies, polyclonalantibodies, monoclonal antibodies, humanized antibodies or chimericantibodies, or a mixture of these.

Examples of antibodies known to specifically bind the MCJ polypeptideset forth herein as SEQ ID NO:1, include, but are not limited tomonoclonal antibodies i) WN.F3, generated from hybridoma N-MCJ 3C1.3F3,which was deposited under ATCC no. #PTA-8135; ii) WN.A12, generated fromhybridoma cell line N-MCJ 3C1.5A12, which was deposited under ATCC no.#PTA-8133; and iii) WN.E4 generated from hybridoma cell line N-MCJ2A2.5E4, which was deposited under. ATCC no. #PTA-8134. (see US PatentPublication US20100129931, published May 27, 2010, which is incorporatedherein by reference). The WN.F3, WN.A12, and WN.E4 antibodies areexamples of antibodies that may be used in methods of the invention asMCJ-modulating compounds. Additional antibodies for use in methods ofthe invention may be produced and tested using art-known methods inconjunction with the disclosure herein and in the disclosure set forthin US Patent Publication US20100129931.

Additional compounds that may be administered in treatment methods ofthe invention include small molecules or chemicals that inhibit MCJpolypeptide activity and small molecules or chemicals that enhance MCJpolypeptide activity. Methods of identifying and testing such smallmolecules and chemicals may include use of art-known library screeningand testing procedures in conjunction with the teaching provided herein.

MCJ polypeptide modulating compounds of the invention may beadministered singly or in combination with one or more additionalcompounds. In some embodiments, a compound of the invention may act in asynergistic manner with one or more other therapeutic agents ortreatments and increase the effectiveness of the one or more therapeuticagents or activities. Thus, for example, administration of a compoundthat inhibits MCJ polypeptide activity in conjunction with a caloricreduction treatment may enhance the efficacy of the caloric reductiontreatment. Thus, an MCJ inhibitor compound may act synergistically toincrease the effectiveness of one or more agents or treatments that canbe administered to treat a metabolic disease or condition.

It will be understood that additional MCJ-modulating compounds can beidentified and used in methods of the invention. For example, candidatecompounds can be can be tested for their ability to increase MCJpolypeptide activity (level and/or function) and their ability to treata metabolic disease or condition using assays and methods presentedherein.

MCJ-modulating compounds of the invention (such as compounds comprisingan MCJ molecule, an anti-MCJ polypeptide antibody or functional fragmentthereof, a small molecule MCJ inhibitor, or a small molecule MCJenhancer, etc.) described herein can be used alone or in conjugates withother molecules such as targeting agents, labeling agents, and/orcytotoxic agents in treatment methods of the invention.

Targeting agents useful according to the methods of the invention arethose that direct a compound of the invention to a specific cell type tobe treated such as liver cells, muscle cells, cardiac cells, circulatorycells, neuronal cells, glial cells, fat cells, skin cells, hematopoieticcells, epithelial cells, sperm, oocytes, muscle cells, adipocytes,kidney cells, hepatocytes, or pancreas cells, or to an organelle such asa mitochondrion. A targeting compound of choice will depend upon thenature of the metabolic disease or condition. In some instances it maybe desirable to target the agent to skeletal muscle, cardiac muscle,kidney, liver, brain, etc. Those of ordinary skill in the art will beaware of and able to select and use suitable targeting agents for use inmethods of the invention. Non-limiting examples of targeting agents formitochondria are Gramicidin S based mitochondrial targeting agents,agents utilizing the carnitine-acylcarnitine translocase system,cytochromes, malate dehydrogenase. Examples of targeting signals thatmay be used in some embodiments of the invention are set forth inDiekert, K., et al., PNAS (1999) vol 96, No. 21, 11752-11757; Addya, S.,et al., J. Cell Biology, (1997) Vol. 139, No. 3, 589-599; Del Gaizo, V.,et al., (2003) Mol. Gen. and Metabol. Vol. 80, 170-180, which areincorporated herein by reference.

Labeling agents may be used in methods of the invention to determine thelocation of MCJ polypeptides in cells and tissues and also, may be usedto assess the cell, tissue, or organelle location of treatment compoundsthat have been administered. Procedures for attaching and utilizinglabeling agents such as enzymatic labels, dyes, radiolabels, etc. arewell known in the art.

Compositions, compounds, and methods of the invention may be enhanced byutilization in combination with other procedures for treating ametabolic disease or condition. In some instances a treatment proceduremay involve administration of another therapeutic agent or treatmentsuch a medicament and/or a behavioral treatment, caloric limitation,surgery, etc. Thus, in some embodiments of the invention, administrationof a compound of the invention (e.g., administration of an anti-MCJantibody or functional fragment thereof, an MCJ polypeptide-encodingnucleic acid, MCJ polypeptide, or a small molecule MCJ enhancer orinhibitor) may be performed in conjunction with therapies for treatingthe metabolic disease or condition such as caloric limitation, increasedphysical activity, surgery, etc. Treatment methods of the invention thatinclude administration of a MCJ-modulating compound can be used at anystages of pre-metabolic disease or condition or when the metabolicdisease or condition is at a later stage, including but not limited toearly-stage, mid-stage, and late-stage of the metabolic disease orcondition, including all times before and after any of these stages.Methods of the invention may also be used for subjects who havepreviously been treated with one or more other medicaments or behavioraltherapy methods that were not successful, were minimally successful,and/or are no longer successful at slowing or stopping progression ofthe metabolic disease or disorder in the subject.

Effective Amounts for Treatments

MCJ-modulating compounds of the invention, (e.g., an anti-MCJ antibodyor functional fragment thereof, an MCJ polypeptide-encoding nucleicacid, MCJ polypeptide, or a small molecule MCJ enhancer or inhibitor,etc.) are administered to the subject in an effective amount fortreating the metabolic disease or condition. An “effective amount fortreating a metabolic disease or condition” is an amount necessary orsufficient to realize a desired biologic effect. For example, aneffective amount of a compound of the invention could be that amountnecessary to (i) slow or halt progression of the disease or condition;or (ii) reverse one or more symptoms of the metabolic disease orcondition. According to some aspects of the invention, an effectiveamount is that amount of a compound of the invention alone or incombination with another medicament or treatment, which when combined orco-administered or administered alone, results in a therapeutic responsein the metabolic disease or condition, either in the prevention or thetreatment of the metabolic disease or condition. The biological effectmay be the amelioration and or absolute elimination of symptomsresulting from the metabolic disease or condition. In anotherembodiment, the biological effect is the complete abrogation of themetabolic disease or condition, as evidenced for example, by adiagnostic test that indicates the subject is free of the disease orcondition.

Typically an effective amount of a compound or drug to increase MCJpolypeptide activity or a compound or drug to decrease MCJ polypeptidewill be determined in clinical trials, establishing an effective dosefor a test population versus a control population in a blind study. Insome embodiments, an effective amount will be that results in a desiredresponse, e.g., an amount that diminishes a metabolic disease orcondition in cells or tissues in a subject with the metabolic disease orcondition. Thus, an effective amount to treat a metabolic disease orcondition characterized by a reduced MCJ polypeptide activity, and/or ametabolic disease or condition for which it is desirable to increase MCJpolypeptide activity, may be the amount that when administered increasesthe amount of MCJ polypeptide activity in the subject to an amount thatthat is above the amount that would occur in the subject or tissuewithout the administration of the composition. Similarly, an effectiveamount to treat a metabolic disease or condition characterized byincreased MCJ polypeptide activity, and/or a metabolic disease orcondition for which it is desirable to decrease MCJ polypeptideactivity, may be the amount that when administered decreases MCJpolypeptide activity in a cell, tissue, and/or subject to an amount thatthat is below the amount that would occur in the subject or tissuewithout the administration of the compound or drug. In the case oftreating a metabolic disease or condition the desired response may bereducing or eliminating one or more symptoms of the metabolic disease orcondition in the cell, tissue, and/or subject. The reduction orelimination may be temporary or may be permanent. The status of themetabolic disease or condition can be monitored using methods ofdetermining MCJ polypeptide activity or levels of nucleic acids thatencode an MCJ polypeptide, etc. In some aspects of the invention, adesired response to treatment of the metabolic disease or condition alsocan be delaying the onset or even preventing the onset of the metabolicdisease or condition.

An effective amount of a compound that modulates (increases ordecreases) MCJ polypeptide activity (also referred to herein as apharmaceutical compound) may also be determined by assessingphysiological effects of administration on a cell or subject, such as adecrease of a metabolic disease or condition following administration.Assays suitable to determine efficacy of a pharmaceutical compound ofthe invention will be known to those skilled in the art and can beemployed for measuring the level of the response to a treatment and anamount of a pharmaceutical compound administered to a subject can bemodified based, at least in part, on such measurements. The amount of atreatment may be varied for example by increasing or decreasing theamount of a therapeutic composition, by changing the therapeuticcomposition administered, by changing the route of administration, bychanging the dosage timing and so on. The effective amount will varywith the particular condition being treated, the age and physicalcondition of the subject being treated; the severity of the condition,the duration of the treatment, the nature of the concurrent therapy (ifany), the specific route of administration, and additional factorswithin the knowledge and expertise of the health practitioner. Forexample, an effective amount may depend upon the degree to which anindividual has abnormally low or abnormally high levels of MCJpolypeptide activity, and/or the desired level of MCJ polypeptideactivity to attain that is effective to treat the metabolic disease orcondition.

The effective amount of a compound of the invention in the treatment ofa metabolic disease or condition or in the reduction of the risk ofdeveloping a metabolic disease or condition may vary depending upon thespecific compound used, the mode of delivery of the compound, andwhether it is used alone or in combination. The effective amount for anyparticular application can also vary depending on such factors as themetabolic disease or condition being treated, the particular compoundbeing administered, the size of the subject, or the severity of themetabolic disease or condition. A skilled artisan can empiricallydetermine the effective amount of a particular compound of the inventionwithout necessitating undue experimentation. Combined with the teachingsprovided herein, by choosing among the various active compounds andweighing factors such as potency, relative bioavailability, patient bodyweight, severity of adverse side-effects and preferred mode ofadministration, an effective prophylactic or therapeutic treatmentregimen can be planned which does not cause substantial toxicity and yetis entirely effective to treat the particular subject.

A pharmaceutical compound dosage may be adjusted by an individual healthcare provider or veterinarian, particularly in the event of anycomplication. A therapeutically effective amount typically varies from0.01 mg/kg to about 1000 mg/kg, from about 0.1 mg/kg to about 200 mg/kg,or from about 0.2 mg/kg to about 20 mg/kg, in one or more doseadministrations daily, for one or more days. The absolute amount willdepend upon a variety of factors including a concurrent treatment, thenumber of doses and the individual subject parameters including age,physical condition, size and weight. These are factors well known tothose of ordinary skill in the art and can be addressed with no morethan routine experimentation. In some embodiments, a maximum dose can beused, that is, the highest safe dose according to sound medicaljudgment.

Multiple doses of compounds of the invention are also contemplated. Insome instances, a compound of the invention, (e.g., an anti-MCJ antibodyor functional fragment thereof, an MCJ polypeptide-encoding nucleicacid, MCJ polypeptide, or a small molecule MCJ enhancer or inhibitor,etc.) can be administered at least daily, every other day, weekly, everyother week, monthly, etc. Doses may be administered once per day or morethan once per day, for example, 2, 3, 4, 5, or more times in one 24 hourperiod.

Pharmaceutical compounds of the invention may be administered alone, incombination with each other, and/or in combination with other drugtherapies, or other treatment regimens that are administered to subjectswith a metabolic disease or condition. Pharmaceutical compositions usedin the foregoing methods preferably are sterile and contain an effectiveamount of a therapeutic compound that will modulate a MCJ polypeptideactivity to a level sufficient to produce the desired response in a unitof weight or volume suitable for administration to a subject.

The doses of a composition to modulate the MCJ polypeptide activity thatis administered to a subject can be chosen in accordance with differentparameters, in particular in accordance with the mode of administrationused and the state of the subject. Other factors include the desiredperiod of treatment. In the event that a response in a subject isinsufficient at the initial doses applied, higher doses (or effectivelyhigher doses by a different, more localized delivery route) may beemployed to the extent that patient tolerance permits.

Administration Methods

A variety of administration routes for a MCJ-modulating compound areavailable. The particular delivery mode selected will depend, of course,upon the particular condition being treated and the dosage required fortherapeutic efficacy. Methods of this invention, generally speaking, maybe practiced using any mode of administration that is medicallyacceptable, meaning any mode that produces effective levels ofprotection without causing clinically unacceptable adverse effects. Insome embodiments of the invention, a compound of the invention may beadministered via an oral, enteral, mucosal, percutaneous, and/orparenteral route. The term “parenteral” includes subcutaneous,intravenous, intramuscular, intraperitoneal, and intrasternal injection,or infusion techniques. Other routes include but are not limited tonasal (e.g., via a gastro-nasal tube), dermal, vaginal, rectal, andsublingual. Delivery routes of the invention may include intrathecal,intraventricular, or intracranial. In some embodiments of the invention,a compound of the invention may be placed within a slow release matrixand administered by placement of the matrix in the subject. In someaspects of the invention, a compound (such as an anti-MCJ antibody orfunctional fragment thereof, an MCJ polypeptide-encoding nucleic acid,MCJ polypeptide, or a small molecule MCJ enhancer or inhibitor, etc.)may be delivered to a subject cell using nanoparticles coated with andelivery agent that targets a specific cell or organelle, a non-limitingexample of which is a mitochondrion.

Compounds of the invention may be administered in formulations, whichmay be administered in pharmaceutically acceptable solutions, which mayroutinely contain pharmaceutically acceptable concentrations of salt,buffering agents, preservatives, compatible carriers, adjuvants, andoptionally other therapeutic ingredients. According to methods of theinvention, the compound may be administered in a pharmaceuticalcomposition. In general, a pharmaceutical composition comprises thecompound of the invention and a pharmaceutically-acceptable carrier.Pharmaceutically-acceptable carriers are well-known to those of ordinaryskill in the art. As used herein, a pharmaceutically-acceptable carriermeans a non-toxic material that does not interfere with theeffectiveness of the biological activity of the active ingredients,e.g., the ability of the compound such as an anti-MCJ antibody orfunctional fragment thereof, an MCJ polypeptide-encoding nucleic acid,MCJ polypeptide, or a small molecule MCJ enhancer or inhibitor, etc. totreat the metabolic disease or condition.

Pharmaceutically acceptable carriers include diluents, fillers, salts,buffers, stabilizers, solubilizers and other materials that arewell-known in the art. Exemplary pharmaceutically acceptable carriersare described in U.S. Pat. No. 5,211,657 and others are known by thoseskilled in the art. Such preparations may routinely contain salt,buffering agents, preservatives, compatible carriers, and optionallyother therapeutic agents. When used in medicine, the salts should bepharmaceutically acceptable, but non-pharmaceutically acceptable saltsmay conveniently be used to prepare pharmaceutically-acceptable saltsthereof and are not excluded from the scope of the invention. Suchpharmacologically and pharmaceutically-acceptable salts include, but arenot limited to, those prepared from the following acids: hydrochloric,hydrobromic, sulfuric, nitric, phosphoric, maleic, acetic, salicylic,citric, formic, malonic, succinic, and the like. Also,pharmaceutically-acceptable salts can be prepared as alkaline metal oralkaline earth salts, such as sodium, potassium or calcium salts.

Compounds of the invention may be administered directly to a tissue. Insome embodiments, the tissue to which the compound is administered is atissue in which the metabolic disease or condition is likely to arise.Direct tissue administration may be achieved by direct injection.Compounds may be administered once, or alternatively they may beadministered in a plurality of administrations. If administered multipletimes, the compounds may be administered via different routes. Forexample, the first (or the first few) administrations may be madedirectly into the affected tissue while later administrations may besystemic.

The compounds, when it is desirable to deliver them systemically, may beformulated for parenteral administration by injection, e.g., by bolusinjection or continuous infusion. Formulations for injection may bepresented in unit dosage form, e.g., in ampoules or in multi-dosecontainers, with or without an added preservative. The compositions maytake such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents.

Preparations for parenteral administration include sterile aqueous ornon-aqueous solutions, suspensions, and emulsions. Examples ofnon-aqueous solvents are propylene glycol, polyethylene glycol,vegetable oils such as olive oil, and injectable organic esters such asethyl oleate. Aqueous carriers include water, alcoholic/aqueoussolutions, emulsions or suspensions, including saline and bufferedmedia. Parenteral vehicles include sodium chloride solution, Ringer'sdextrose, dextrose and sodium chloride, lactated Ringer's, or fixedoils. Intravenous vehicles include fluid and nutrient replenishers,electrolyte replenishers (such as those based on Ringer's dextrose), andthe like. Preservatives and other additives may also be present such as,for example, antimicrobials, antioxidants, chelating agents, and inertgases and the like. Lower doses will result from other forms ofadministration, such as intravenous administration. In the event that aresponse in a subject is insufficient at the initial doses applied,higher doses (or effectively higher doses by a different, more localizeddelivery route) may be employed to the extent that patient tolerancepermits. Multiple doses per day may be used as needed to achieveappropriate systemic or local levels of compounds.

In yet other embodiments, a delivery vehicle is a biocompatiblemicroparticle or implant that is suitable for implantation into themammalian recipient. Exemplary bioerodible implants that are useful inaccordance with this method are described in PCT Publication No. WO95/24929 (incorporated by reference herein), which describes abiocompatible, biodegradable polymeric matrix for containing abiological macromolecule. Such delivery means are well known in the artand can be used to achieve sustained release of a compound of theinvention in a subject, and may be selected not to degrade, but rather,to release by diffusion over an extended period of time.

Both non-biodegradable and biodegradable polymeric matrices can be usedto deliver the compounds of the invention to the subject. In someembodiments, a matrix may be biodegradable. Matrix polymers may benatural or synthetic polymers. A polymer can be selected based on theperiod of time over which release is desired, generally in the order ofa few hours to a year or longer. Typically, release over a periodranging from between a few hours and three to twelve months can be used.The polymer optionally is in the form of a hydrogel that can absorb upto about 90% of its weight in water and further, optionally iscross-linked with multivalent ions or other polymers.

In general, compounds of the invention may be delivered using thebioerodible implant by way of diffusion, or by degradation of thepolymeric matrix. Exemplary synthetic polymers for such use are wellknown in the art. Biodegradable polymers and non-biodegradable polymerscan be used for delivery of compounds of the invention using art-knownmethods. Bioadhesive polymers such as bioerodible hydrogels (see H. S.Sawhney, C. P. Pathak and J. A. Hubell in Macromolecules, 1993, 26,581-587, the teachings of which are incorporated herein) may also beused to deliver compounds of the invention for treatment. Additionalsuitable delivery systems can include time-release, delayed release orsustained release delivery systems. Such systems can avoid repeatedadministrations of the compound, increasing convenience to the subjectand the physician. Many types of release delivery systems are availableand known to those of ordinary skill in the art. (See for example: U.S.Pat. Nos. 5,075,109; 4,452,775; 4,675,189; 5,736,152; 3,854,480;5,133,974; and 5,407,686 (the teaching of each of which is incorporatedherein by reference). In addition, pump-based hardware delivery systemscan be used, some of which are adapted for implantation.

Use of a long-term sustained release implant may be particularlysuitable for prophylactic treatment of subjects and for subjects at riskof developing a recurrent metabolic disease or condition. Long-termrelease, as used herein, means that the implant is constructed andarranged to delivery therapeutic levels of the active ingredient for atleast 30 days, 60 days, 90 days or longer. Long-term sustained releaseimplants are well-known to those of ordinary skill in the art andinclude some of the release systems described above.

Therapeutic formulations of compounds of the invention may be preparedfor storage by mixing the compound having the desired degree of puritywith optional pharmaceutically acceptable carriers, excipients orstabilizers [Remington's Pharmaceutical Sciences 21^(st) edition,(2006)], in the form of lyophilized formulations or aqueous solutions.Acceptable carriers, excipients, or stabilizers are nontoxic torecipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

Detection Assays and Diagnostics

Certain aspects of the invention include methods to assess the status ofa metabolic disease or condition characterized by abnormal MCJpolypeptide activity. Such methods may include determining a level orfunction of an MCJ polypeptide and comparing the determined level orfunction with a control level or function. In some embodiments of theinvention methods include use of antibodies or antigen-binding fragmentsthat specifically bind MCJ polypeptide in assays to detect a level of anMCJ polypeptide, or a nucleic acid that encodes an MCJ polypeptide in acell and/or subject. Such assays may include obtaining a biologicalsample from a subject, determining the level of an MCJ molecule in thebiological sample, and comparing the level to a control level. As usedherein a biological sample may be an in vitro biological sample, or maya sample that is detected (e.g., obtained) in vivo. As used herein, abiological sample may be a cell sample, tissue sample, blood sample,bodily fluid sample, subcellular sample, etc. A biological sample mayinclude cells, tissues, or organelles and may include cell types such asbut not limited to: liver cells, muscle cells, cardiac cells,circulatory cells, neuronal cells, glial cells, fat cells, skin cells,hematopoietic cells, epithelial cells, sperm, oocytes, muscle cells,adipocytes, kidney cells, hepatocytes, pancreas cells, etc. In someembodiments of the invention, a biological sample may comprise one ormore cancer cells. In certain embodiments of the invention, a biologicalsample does not comprise a cancer cell.

Assays to assess a metabolic disease or condition characterized byabnormal MCJ polypeptide activity may include but are not limited to (1)characterizing the impact of MCJ polypeptide activity on treatment of ametabolic disease or condition in a subject; (2) evaluating a treatmentto alter MCJ polypeptide activity (e.g., level and/or function) in asubject; (3) selecting a treatment for a metabolic disease or conditionbased at least in part on the determined MCJ polypeptide activity incells of the subject; and (4) determining the status of a metabolicdisease or condition in a subject. Thus, subjects can be characterized,treatment regimens can be monitored, treatments can be selected anddiseases status can be better understood using embodiments of methods ofthe present invention. For example, the antibodies or antigen-bindingfragments thereof of the invention and other methods of the inventionare useful in to measure or determine MCJ-polypeptide activity in a celland/or subject, which may be a direct indicator of a metabolic diseaseor condition in the cell and/or subject.

The invention, in some aspects, includes various assays to determineactivity of an MCJ polypeptide. Methods of the invention that are usefulto determine MCJ polypeptide activity (levels and/or function of the MCJpolypeptide) in cells, tissues, subjects, and samples (e.g., fromsubjects, in culture, etc.), include, but are not limited to: bindingassays, such as using antibodies or antigen-binding fragments thereofthat bind specifically to an MCJ polypeptide; gel electrophoresis; massspectrometry; NMR; and the like. Immunoassays may be used according tothe invention including, but not limited to, sandwich-type assays,competitive binding assays, one-step direct tests and two-step testssuch as those routinely used in the art. Assessment of binding ofantibodies that specifically bind MCJ polypeptide may also be done invivo—in living subjects using art-known detectable labels and suitablein vivo methods.

Methods and assays of the invention (e.g. binding assays, gelelectrophoresis; mass spectrometry; NMR; and the like) may be used todetermine changes in MCJ polypeptide activity in a subject or cellsample (e.g., cell culture) over time. This allows monitoring of MCJpolypeptide activity in a subject who is to undergo treatment for ametabolic disease or condition and also enables to monitoring in asubject who is currently undergoing therapy for a metabolic disease orcondition. Thus, methods of the invention may be used to diagnose orassess a metabolic disease or condition in a subject and may also beused to assess the efficacy of a therapeutic treatment of a metabolicdisease or condition for assessment of the activity of an MCJpolypeptide in a subject at various time points. For example, asubject's MCJ polypeptide activity can be determined prior to the startof a therapeutic regimen (either prophylactic or as a treatment of ametabolic disease or condition), during the treatment regimen and/orafter a treatment regimen, thus providing information on the status ofthe metabolic disease or condition in the subject.

Assessment of efficacy of candidate MCJ-modulating compounds to increaseor decrease expression of MCJ polypeptide-encoding nucleic acid or anMCJ polypeptide in a cell or tissue may also be done using assays of theinvention in cells from culture—e.g., as screening assays to assesscandidate MCJ-modulating compounds to modulate MCJ polypeptide activity.MCJ-modulating compounds that alter MCJ polypeptide activity in a cell,tissue, or subject may be used in the treatment of a metabolic diseaseor condition or as a pretreatment for a metabolic disease or condition(e.g., to prepare a cell or subject for subsequent treatment).

It will be understood that a therapeutic regimen may be eitherprophylactic or a treatment of a metabolic disease or condition in asubject. The invention in some aspects provides methods that may be usedto monitor a subject's response to prophylactic therapy and/or treatmentfor a metabolic disease or condition provided to a subject. Methods ofthe invention (e.g. binding assays, gel electrophoresis; massspectrometry; NMR; and the like) may also be useful to monitor theonset, progression, or regression of metabolic disease or condition in asubject at risk of developing the metabolic disease or condition. MCJpolypeptide activity may be determined in two, three, four, or morebiological samples obtained from a subject at separate times. The MCJpolypeptide activity determined in the samples may be compared andchanges in the activity over time may be used to assess the status andstage of a metabolic disease or condition in the subject (or in a cellor tissue sample) and/or the effect of a treatment strategy on themetabolic disease or condition in a subject (or a cell or tissuesample). Antibodies or fragments thereof that specifically bind MCJpolypeptide can be used to obtain useful prognostic information byproviding an indicator of a metabolic disease or condition and can beused to select a therapy for the subject, for example, to select a drugtherapy, behavioral therapy, surgical therapy, etc.

Assays described herein may include determining MCJ polypeptideactivity, including but not limited to determining levels of nucleicacids that encode MCJ polypeptides and/determining levels of MCJpolypeptides in cells, tissues, and subjects. Levels of MCJpolypeptide-encoding nucleic acids and polypeptides can be determined ina number of ways when carrying out the various methods of the invention.In some embodiments of the invention, a level of MCJpolypeptide-encoding nucleic acid or polypeptide is measured in relationto a control level of MCJ-polypeptide-encoding nucleic acid orpolypeptide, respectively, in a cell, tissue, or subject. One possiblemeasurement of the level of MCJ polypeptide-encoding nucleic acid orpolypeptide is a measurement of absolute levels ofMCJ-polypeptide-encoding nucleic acid or polypeptide. This could beexpressed, for example, in MCJ-polypeptide-encoding nucleic acid orpolypeptide per unit of cells or tissue. Another measurement of a levelof MCJ polypeptide-encoding nucleic acid or polypeptide is a measurementof the change in the level of MCJ-polypeptide-encoding nucleic acid orpolypeptide over time. This may be expressed in an absolute amount ormay be expressed in terms of a percentage increase or decrease overtime. Antibodies or antigen-binding fragments or other compounds thatspecifically bind MCJ polypeptide or a nucleic acid that encodes MCJpolypeptide may be used in diagnostic methods alone or in conjunctionwith certain antibodies or binding compounds already known in the art.Known antibodies may include antibodies that specifically bind to otherproteins that are associated with metabolic diseases or conditions orother cell marker proteins that may be used to quantitate the level ofMCJ polypeptide-encoding nucleic acid or polypeptide per unit of cells,etc.

As mentioned above, it is also possible to use a compound of theinvention (e.g., an anti-MCJ antibody or functional fragment thereof, anMCJ polypeptide-encoding nucleic acid, MCJ polypeptide, or a smallmolecule MCJ enhancer or inhibitor, etc.) to characterize MCJpolypeptide activity by monitoring changes in the MCJ polypeptideactivity over time. For example, in certain metabolic diseases andconditions, an increase in MCJ polypeptide activity correlates withincreased likelihood of a metabolic disease or condition in cells and/ortissues and in certain metabolic diseases and conditions, a decrease inMCJ polypeptide activity correlates with increased likelihood of ametabolic disease or condition in cells and/or tissues. In addition,certain metabolic diseases and conditions may not be directlycharacterized by abnormal MCJ polypeptide activity, but can be treatedby modulating (in some increasing and in other cases decreasing) MCJpolypeptide activity. In each type of metabolic disease or condition itmay be desirable to assess MCJ polypeptide activity in a treated cell,tissue, or subject.

Accordingly one can monitor MCJ polypeptide activity levels over time todetermine if there is a change in status of a metabolic disease orcondition in a subject or in a cell culture. Changes in MCJ polypeptideactivity such as an increase or decrease in MCJ polypeptide activitythat is greater than 0.1% of the baseline activity level, a previousactivity level, or a control activity level may be an indicator ofefficacy of, or need for, a treatment of the invention to decrease orincrease, respectively, MCJ polypeptide activity.

In some embodiments of the invention, an increase in an MCJ polypeptideactivity in a cell or tissue may be an increase greater than 0.2%,greater than 0.5%, greater than 1.0%, 2.0%, 3.0%, 4.0%, 5.0%, 7.0%, 10%,15%, 20%, 25%, 30%, 40%, 50%, or more from a previous MCJ polypeptideactivity in the subject. In some embodiments of the invention, adecrease in an MCJ polypeptide activity level in a cell or tissue, maybe a decrease of more than 0.2%, more than 0.5%, more than 1.0%, 2.0%,3.0%, 4.0%, 5.0%, 7.0%, 10%, 15%, 20%, 25%, 30%, 40%, 50%, or more.Increases or decreases in the MCJ polypeptide activity determinationsover time may indicate a change in the status of a metabolic disease orcondition in a sample or subject, efficacy of a treatment of a metabolicdisease or condition, or the attainment of a desirable level of MCJpolypeptide activity in a metabolic disease or condition.

As a determination of the status of a metabolic disease or condition,methods of the invention may be used to determine MCJ polypeptideactivity in two or more samples, taken at different times, and tocompare the determinations to each other and/or with control MCJpolypeptide activity levels. Such comparisons may be used to determinethe status of a metabolic disease or condition in a subject and allowsevaluation of a treatment of a metabolic disease or condition.Comparison of a subject's MCJ polypeptide activity measured inbiological samples obtained from the subject at different times and/oron different days can be used as a measure of the effectiveness of anytreatment for the metabolic disease or condition in the subject. Thoseof ordinary skill in the art will recognize that efficacy of treatmentmethods and candidate therapeutics can be tested in vitro by assessingany change in MCJ polypeptide activity of a cell that occurs in responseto contacting the cell with a candidate agent for treatment of ametabolic disease or condition or with a candidate agent for themodulation of MCJ polypeptide activity.

As will be appreciated by those of ordinary skill in the art, theevaluation of a treatment also may be based upon an evaluation of thesymptoms or clinical end-points of a metabolic disease or condition andsuch evaluations can be used in conjunction with methods of theinvention to assess the status of a metabolic disease or conditionand/or the efficacy of a treatment of a metabolic disease or condition.

Kits

Also within the scope of the invention are kits that comprisecompositions of the invention and instructions for use. Kits of theinvention may include one or more of a compound such as an anti-MCJantibody or functional fragment thereof, an MCJ polypeptide-encodingnucleic acid, MCJ polypeptide, or a small molecule MCJ enhancer orinhibitor, etc., which may be used to treat a metabolic disease orcondition or in some aspects of the invention to diagnose or monitor ametabolic disease or condition. Kits containing compounds such as ananti-MCJ antibody or functional fragment thereof, an MCJpolypeptide-encoding nucleic acid, MCJ polypeptide, or a small moleculeMCJ enhancer or inhibitor, etc. can be prepared for treatment methods,in vitro diagnosis, prognosis and/or monitoring the level of MCJpolypeptide activity in cells, tissues, and/or subjects using anysuitable histological, cytological, serological or other method.Components of kits of the invention may be packaged either in aqueousmedium or in lyophilized form. A kit of the invention may comprise acarrier being compartmentalized to receive in close confinement thereinone or more container means or series of container means such as testtubes, vials, flasks, bottles, syringes, or the like. A first containermeans or series of container means may contain one or more compoundssuch as compound an anti-MCJ antibody or functional fragment thereof, anMCJ polypeptide-encoding nucleic acid, MCJ polypeptide, or a smallmolecule MCJ enhancer or inhibitor, etc. A second container means orseries of container means may contain a targeting label or linker-labelintermediate capable delivering a compound to a cell or tissue.

A kit of the invention may also include instructions. Instructionstypically will be in written form and will provide guidance forcarrying-out the assay or treatment embodied by the kit and for making adetermination based upon that assay or treatment.

Methods to Identify Candidate Compounds

Certain aspects of the invention include methods of identifying and/orscreening candidate agents that modulate MCJ polypeptide activity incells, tissues, and/or subjects. Methods can include mixing thecandidate agent with cells or tissues or in a subject and determine theMCJ polypeptide activity before and after contact with the candidateagent. An increase in the amount of MCJ polypeptide activity incomparison to a suitable control is indicative of an agent capable ofincreasing the level of MCJ. A decrease in the amount of MCJ polypeptideactivity in comparison to a suitable control is indicative of an agentcapable of decreasing the level of MCJ.

An assay mixture useful to assess a treatments candidate for a metabolicdisease or disorder comprises a candidate agent. The candidate agent maybe an antibody, a small organic compound, small molecule, polypeptide,nucleic acid, etc., and accordingly can be selected from combinatorialantibody libraries, combinatorial protein libraries, small organicmolecule libraries, or any other suitable source. Typically, a pluralityof reaction mixtures is run in parallel with different agentconcentrations to obtain a different response to the variousconcentrations. Typically, one of these concentrations serves as anegative control, i.e., at zero concentration of agent or at aconcentration of agent below the limits of assay detection.

Candidate agents that can be tested to determine whether they may beuseful to modulate MCJ polypeptide activity, and may be useful to treata metabolic disease or condition, may encompass numerous chemicalclasses, nucleic acids, proteins, etc. In some embodiments of theinvention the candidate agents are small organic compounds, i.e., thosehaving a molecular weight of more than 50 yet less than about 2500,preferably less than about 1000 and, more preferably, less than about500. Candidate agents comprise functional chemical groups necessary forstructural interactions with polypeptides and/or nucleic acids, andtypically include at least an amine, carbonyl, hydroxyl, or carboxylgroup, preferably at least two of the functional chemical groups andmore preferably at least three of the functional chemical groups. Thecandidate agents can comprise cyclic carbon or heterocyclic structureand/or aromatic or polyaromatic structures substituted with one or moreof the above-identified functional groups. Candidate agents also can bebiomolecules such as polypeptides, saccharides, fatty acids, sterols,isoprenoids, purines, pyrimidines, derivatives or structural analogs ofthe above, or combinations thereof and the like.

Candidate agents are obtained from a wide variety of sources includinglibraries of synthetic or natural compounds. For example, numerous meansare available for random and directed synthesis of a wide variety oforganic compounds and biomolecules, including expression of randomizedoligonucleotides, synthetic organic combinatorial libraries, phagedisplay libraries of random or non-random polypeptides, combinatoriallibraries of proteins or antibodies, and the like. Alternatively,libraries of natural compounds in the form of bacterial, fungal, plant,and animal extracts are available or readily produced. Additionally,natural and synthetically produced libraries and compounds can bereadily be modified through conventional chemical, physical, andbiochemical means. Further, known agents may be subjected to directed orrandom chemical modifications such as acylation, alkylation,esterification, amidification, etc. to produce structural analogs of theagents.

A variety of other reagents also can be included in an assay mixture totest a candidate compound. These may include reagents such as salts,buffers, neutral proteins (e.g., albumin), detergents, etc., which maybe used to facilitate optimal protein-protein and/or protein-agentbinding. Such a reagent may also reduce non-specific or backgroundinteractions of the reaction components. Other reagents that improve theefficiency of the assay such as protease inhibitors, nucleaseinhibitors, antimicrobial agents, and the like may also be used. Theorder of addition of components, incubation temperature, time ofincubation, and other parameters of the assay may be readily determined.Such experimentation merely involves optimization of the assayparameters, not the fundamental composition of the assay. Incubationtemperatures typically are between 4° C. and 40° C. Incubation times maybe minimized to facilitate rapid, high throughput screening, andtypically are between 0.1 and 10 hours. After incubation, variables suchas the presence or absence of MCJ polypeptides and MCJ polypeptideactivity can be detected by any convenient method available to the user.For example, MCJ polypeptide activity can be determined through themeasure of a detectable label using standard methods and as describedherein.

The following examples are provided to illustrate specific instances ofthe practice of the present invention and are not intended to limit thescope of the invention. As will be apparent to one of ordinary skill inthe art, the present invention will find application in a variety ofcompositions and methods.

EXAMPLES Example 1

Methods

Mice.

C57Bl/6J mice were purchased from Jackson Laboratories (Bar Harbor,Me.). MCJ targeted embryonic stem cells (RRN 226) were obtained fromBaygenomics [University of California San Francisco (UCSF), SanFrancisco, Calif.]. The gene trapped ES cells were injected in C57B1/6Jblastocysts and implanted in pseudo-pregnant females at the Universityof Vermont Transgenic Mouse Facility. Six male chimeras were obtainedwith 5 showing more than 95% chimerism. Chimeras were crossed withC57Bl/6J females and all of them led to germline transmission (100%).Mice were further backcrossed with C57Bl/6 for at least 7 generations.The heterozygous males and females were crossed for the generation ofMCJ homozygous knockout. MCJ KO mice were also crossed with thepreviously described OT-I TCR transgenic mice (Hogquist et al., 1994Cell 76, 17-27) and MMTV-PyMT mice (Guy et al., 1992 Mol Cell Biol 12,954-961). Mice were used between 10 and 14 weeks of age. For thestarvation studies, mice were kept with water but not food for 36 h.Blood, liver, kidney and brown fat were harvested. For liver cholesterolaccumulation studies, the mice were kept on a high-cholesterol diet(Harlan Keklad T D. 902221) for 4 weeks as described in see Plavinskaya,T. et al., Pulm Pharmacol Ther. Effects of acute and chronic low densitylipoprotein exposure on neutrophil function, Epub 2012 Oct. 17. All micewere housed under sterile conditions at the animal care facility at theUniversity of Vermont. The procedures were approved by the University ofVermont Institutional Animal Care and Use Committee.

Cell Preparation, Culture Conditions, Proliferation and Reagents.

CD8 T cells and CD4 T cells were purified from spleen and lymph nodes bynegative selection as previously described (Auphan et al., 1998 JImmunol 160, 4810-4821; Conze et al., 2002 J Exp Med 195, 811-823), andby positive selection using the MACS system as recommended by themanufacturer (Miltenyi Biotec, Cambridge, Mass.). Purified T cells werestimulated with plate-bound anti-CD3 (2C11) (5 μg/ml) and solubleanti-CD28 (1 μg/ml) (BD Biosciences, Sparks, Md.) mAbs. Proliferationassays were performed using purified CD8 T cells (10⁵ cells/well)activated with anti-CD3 and anti-CD28 and labeled with [³H]-thymidine aspreviously described (Conze et al., 2002 J Exp Med 195, 811-823). MCF7,MCF7/siMCJ, and 293T cell lines were maintained as previously described(Hatle et al., 2007 Mol Cell Biol 27, 2952-2966). Rotenone (used at 10μM) was purchased from Sigma Aldrich (St. Louis, Mo.). Transfection of293T cells by calcium phosphate was performed as previously described(Hatle et al., 2007 Mol Cell Biol 27, 2952-2966).

Northern Blot Analysis.

Human and Mouse multiple tissue Northern blots (MTN) were purchased fromClontech Laboratories, Inc. (Mountain View, Calif.) and containednormalized levels of PolyA RNA from different tissues. Radiolabeling ofboth mouse and human MCJ probes was performed as described (Rincón etal., 1988 Eur J Immunol November; 18(11):1791-6) and Northern blotanalysis was done as per the manufacturer's instructions.

Southern Blot Analysis.

10 μg of tail genomic DNA digested with NcoI were used. DNA fragmentswere separated in an agarose gel, transferred onto a Hybond™ nylonmembrane and radiolabeled probed with a PCR amplified region from MCJintron 1 [5′-gtggg ggtgtctgtgaagtagttt-3′ (SEQ ID NO:6) and5′-ctgggatttaaggagttcacaa-3′ (SEQ ID NO:7)].

RNA Isolation and RT-PCR.

Total RNA was isolated using the Qiagen mini RNeasy® kit, as recommendedby the manufacturer (Qiagen, Valencia, Calif.). The first strand cDNAwas obtained by reverse transcription as described previously (Hatle etal., 2007 Mol Cell Biol 27, 2952-2966). cDNA was used to detect mouseHPRT, MCJ, beta 2 microglobulin by conventional PCR or real-time RT-PCR.For the real-time RT-PCR analysis (Applied Biosystems™, Carlsbad,Calif.) the following primers/probe designed set was used for mouse MCJ[Forward 5′-ccg aat acc tgc ctc ctt ctg-3′ (SEQ ID NO:8), Reverse 5′-acacag cgg gga gaa ggt t-3′ (SEQ ID NO:9), Probe 5′-cca aag gtc gga cgc cgacat c-3′ (SEQ ID NO:10)]. The relative values were determined by thecomparative CT analysis method using hypoxanthinephosphoribosyltransferase (HPRT) or β₂-microglobulin as housekeepinggenes. Conventional RT-PCR amplification of MCJ was done using thefollowing primers [5′-aag taa tca cgg caa cag caa gg-3′ (SEQ ID NO:11)and 5′-aat aaa agc ctg gca gcc ttg c-3′ (SEQ ID NO:12)].

Western Blot Analysis.

Whole cell extracts were prepared in triton lysis buffer as previouslydescribed (Hatle et al., 2007 Mol Cell Biol 27, 2952-2966).Mitochondrial and cytosolic extracts were purified using the CellFractionation Kit Standard (MitoSciences®, Eugene, Oreg.) for CD8 Tcells and MCF7 cells or the Mitochondrial Fractionation Kit (ActiveMotif®, Carlsbad, Calif.) for heart, tumor, and other tissues.Mitochondrial extracts were solubilized with either lauryl maltoside(1%) or digitonin (2%) when specified. Isolation of the mitochondrialinner membrane fraction was performed as previously described (see DaCruz, S. et al., 2003 J. Biol. Chem. 278:41566-41571) using purifiedmitochondrial extracts from the heart. Western blot analyses wereperformed as previously described (Hatle et al., 2007 Mol Cell Biol 27,2952-2966). The anti-mouse MCJ rabbit polyclonal was generated byimmunization with the N-terminal peptide (35 aa) of mouse MCJ followingaffinity immunopurification (Cocalico Biologicals, Inc.™, Reamstown,Pa.). The anti-human MCJ mouse monoclonal Ab has already been described(Hatle et al., 2007 Mol Cell Biol 27, 2952-2966). Other antibodies usedwere: antiactin, anti-glyceraldehyde-3-phosphate dehydrogenase(anti-GAPDH), anti-rabbit IgG, anti-goat IgG, anti-mouse IgG (Santa CruzBiotechnology, Santa Cruz, Calif.), anti-CoxIV (Cell SignalingTechnology®, Danvers, Mass.), anti-NDUFA9 and anti-NDUFS3(MitoSciences®, Eugene, Oreg.) and complex III Corel protein(MitoSciences); anti-glycogen synthase (Cell Signaling Technology®anti-phosphoenolpyruvate carboxykinase (anti-PEPCK) (Santa CruzBiotechnology, Dallas, Tex.); calreticulin (Enzo Life Science,Farmingdale, N.Y.); and sintaxin 11 (BD Bioscience, San Jose, Calif.).LumiGLO® chemiluminescent substrate system (KPL, Gaithersburg, Md.) wasused to visualize the proteins. Immunoprecipitation of Complex I wasperformed using mitochondria extracts generated as described above andthe Complex I Immunocapture monoclonal antibody (MitoSciences Eugene,Oreg.) as recommended by the manufacturer (MitoSciences °, Eugene,Oreg.). Immunoprecipitations of complex I and complex III were performedusing mitochondrial extracts generated as described above andsolubilized with maltoside (1%) as the detergent and the complex I orcomplex III Immunocapture monoclonal antibody (MitoSciences) asrecommended by the manufacturer. The immunoprecipitates were thenexamined for the presence of MCJ, or specific subunits were examined forcomplex I, III, or IV subunits by Western blotting. Immunoprecipitateswere then examined for the presence of MCJ and Complex I and IIIsubunits by Western blot analysis.

Flow Cytometry Analysis.

Cell surface staining was performed using directly labeled antibodiesfor CD4, CD8, Thy1.1, Thy1.2, Vα2 [BD Biosciences (Sparks, Md.) andBioLegend® (San Diego, Calif.)]. Staining for cell viability wasperformed using the UV-Blue dye [Molecular Probes®, Inc, LifeTechnologies, (Beverly, Mass.)] as recommended by the manufacturer.Mitochondrial membrane potential analysis was performed by staining withTetramethylrhodamine, Ethyl Ester, Perchlorate (TMRE) [MolecularProbes®, Inc, Life Technologies, (Beverly, Mass.)] (10 μM) for 20 min at37° C. as recommended by the manufacturer. Mitochondrial ROS analysiswas performed by staining with Mito-SOX™-Red (Invitrogen®, LifeTechnologies, Beverly, Mass.) (2.5 μM) for 10 min at 37° C. asrecommended by the manufacturer. All samples were examined by flowcytometry analysis using an LSRII flow cytometer (BD Biosciences,Sparks, Md.) and FLOWJO® software (Flowjo, Ashland, Oreg.).

Cell sorting purification of peripheral CD4 T cells, CD8 T cells and Bcells for MCJ expression was performed by staining with the Abs for CD4,CD8 and B220 (B cell marker), and the Aria Flow Cell Sorter (BDBiosciences, Sparks, Md.).

Confocal Microscopy Analysis.

Cell preparation and immunostaining of transfected 293T cells forconfocal microscopy analysis were performed as we previously described(Hatle et al., 2007 Mol Cell Biol 27, 2952-2966). Mito Tracker® andTOPRO® [Molecular Probes®, Life Technologies, (Beverly, Mass.)] wereused as markers for mitochondria and nuclei, respectively. The anti-HAtag Ab (Cell Signaling Technology®, Danvers, Mass.) was used fordetection of the HA-tagged MCJ followed by the anti-rabbit secondary Ab[Molecular Probes®, Life Technologies, (Beverly, Mass.)]). Samples wereexamined by confocal microscopy using a Zeiss LSM 510 META confocallaser scanning imaging system (Carl Zeiss Microimaging, Thornwood,N.Y.).

Immunoelectron Microscopy Analysis.

Immunoelectron microscopy analysis for MCJ was performed as wepreviously described (Hatle et al., 2007 Mol Cell Biol 27, 2952-2966)using fixed embedded preparations of MCJ-transfected 293T cells, freshlyisolated CD8 T cells or heart tissue. The anti-mouse MCJ rabbitpolyclonal Ab was used for detection of MCJ.

Histology and Sera Biochemistry Analysis.

For histological analysis, liver, kidneys, brown fat and mammary tumorswere harvested, fixed in formalin and paraffin embedded. Tissue sectionsfrom paraffin embedded blocks were stained with H&E according to routineprocedures. Images were obtained by the Olympus® BX50 light microscopewith a Magnafire® digital camera from Optronics® (Goleta, Calif.) orwith the EVOS® XL Core microscope (AMG, Life Technologies, Grand Island,N.Y.). For analysis of lipid accumulation in the liver, freshlyharvested livers were frozen in OCT and frozen sections were stained forOil Red O. For histological analysis of glycogen, periodic acid-Schiff(PAS) staining was performed in paraffin-embedded liver and kidneysections. The levels of glycogen in liver extracts (corresponding to 1mg) were determined using the Glucose (HK) Assay kit, as recommended bythe manufacturer (Sigma Aldrich, Saint Louis, Mo.). Levels oftriglycerides in serum were determined using the Triglyceridecolorimetric kit (BioVision, Milpitas, Calif.) and ketone body kit(Cayman Chemicals, Ann Arbor, Mich.) as recommended by themanufacturers. The levels of free fatty acids (FFA) in serum and liverextracts were determined using the Free Fatty Acid quantification kit(Cayman Chemical®, Ann Arbor, Mich.) and/or free fatty acidquantification kit (BioVision, Milpitas, Calif.) as recommended by themanufacturers. The levels of cholesterol in the liver extracts(corresponding to 1 mg) were determined using the Abcam kit (Abcam,Cambridge, Mass.). Glucose levels in blood were determined using theglucose-monitoring system (LifeScan, Milpitas, Calif.).

Complex I Activity.

Analysis of complex I activity was performed using mitochondrialextracts generated following the protocol for the purification ofComplex I (MitoSciences®, Eugene, Oreg.). The activity assay using theComplex I Enzyme Activity Microplate assay kit from MitoSciences®(Eugene, Oreg.) and the protocol recommended by the manufacturer.Mitochondrial extracts were normalized among samples and a total of 1 μg(heart), 5-20 μg (MCF7 and MCF7/siMCJ cells), or 1.8 μg (T cells) wereused for the assay.

Intracellular ATP Levels Determination.

The levels of intracellular ATP were determined using 104 cells (CD8 Tcells, MCF7 cells, 293T cells) per sample and the ATPlite® LuminescenceATP Detection Assay System (PerkinElmer®, Waltham, Mass.) following therecommendations from the manufacturer and a TD-20/20 luminometer (TurnerBiosystems, Promega®, Madison, Wis.). For analysis of ATP levels inmammary tumor tissue, cytosolic and mitochondria extracts were generatedas described above and an amount equivalent to 10 μg of protein wasassayed for ATP content using the ATPlite® kit (PerkinElmer®, Waltham,Mass.).

It has now been shown that MCJ resides in the mitochondria where itserves as a negative regulator of Complex I of the mitochondrialrespiratory chain. Loss of MCJ leads to increased Complex I activity,hyperpolarization of mitochondria and increased generation of ATP.Enhanced mitochondrial oxidative phosphorylation as a source of energyin the absence of MCJ accelerates fatty acid oxidation and preventsaccumulation of lipids in the liver during starvation. In addition, MCJdeficiency also delayed mammary tumor growth correlating with theinability to attenuate mitochondrial function. Thus, MCJ is an essentialnegative regulator of mitochondrial metabolism.

Results

Identification of Murine MCJ/DnaJC15 Reveals a Conserved TissueExpression Pattern with the Human Ortholog.

MCJ/DnaJC15 expression has previously been examined in human ovarian andbreast cancer cells (Hatle et al., 2007 Mol Cell Biol 27, 2952-2966;Lindsey et al., 2005 Int J Cancer January 15; 118(2):346-52; Shridhar etal., 2001 Cancer Res 61, 4258-4265; Strathdee et al., 2004Carcinogenesis 25, 693-701). It was previously reported that MCJoriginated in vertebrates where it is highly conserved (Hatle et al.,2007 Mol Cell Biol 27, 2952-2966), but no studies have reported itsmurine counterpart. Comparative analysis of human and mouse MCJ proteinsequences showed an overall 75% identity (FIG. 1A), with nearlyidentical transmembrane and C-terminal DnaJ domain regions, as well as ahighly conserved (57%) Nterminal region (1-35 amino acids). To identifythe tissue expression pattern of murine MCJ gene, Northern blot analysiswas performed using methods outlined above. MCJ mRNA was highly abundantin the heart, followed by liver and kidney (FIG. 1B). Although MCJ isexpressed in some human cancer cells, the specific distribution of MCJexpression in normal human tissues remained unknown. Northern blotanalysis of non-malignant human tissues showed a distribution of humanmcj gene expression similar to the murine mcj gene (FIG. 1C). Previousmicroarray analyses performed using CD8 T cells indicated that MCJ wasalso present in this immune cell type (data not shown). To furtherinvestigate the expression of MCJ in the different populations of theimmune system, CD4 and CD8 T cells, as well as B cells were isolatedfrom mouse spleen and lymph nodes and performed real time reversetranscriptase (RT) PCR. Interestingly, mcj gene expression was very highin CD8 T cells, but almost undetectable in CD4 T cells and B cells (FIG.1D).

An antibody (Ab) that specifically recognizes the N-terminal region ofmouse MCJ was generated, as confirmed by Western blot analysis of 293Tcells transfected with murine MCJ (FIG. 1E). The expression ofendogenous MCJ protein in mouse tissues was examined by Western blotanalysis using this Ab. Consistent with the mRNA expression analysis,MCJ protein was present in heart, liver and kidney, but almostundetectable in lungs (FIG. 1F). MCJ protein also was abundant in CD8 Tcells, but low in CD4 T cells (FIG. 1G). Thus, it was identified thatMCJ/DnaJC15 has a restricted tissue and cellular distribution.

MCJ/DnaJC15 is a Novel Mitochondrial Resident Cochaperone.

Phylogenic analyses have shown that the ancestor of MCJ is Tim14 presentin yeast (Mokranjac et al., 2003 Embo J 22, 4945-4956). Tim14 islocalized in the mitochondrial inner membrane and is a component of theTIM23 translocase (Mokranjac et al., 2005 J Biol Chem 280, 31608-31614).It has previously been reported that MCJ did not localize inmitochondria based on overexpression analysis of MCJ in 293T cells (thatdo not express endogenous MCJ) and Mito Tracker® staining using confocalmicroscopy (Hatle et al., 2007 Mol Cell Biol 27, 2952-2966). Inaddition, no MCJ immunostaining could be found in well-definedmitochondria by immunoelectron microscopy (IEM) in transfected 293Tcells, but MCJ immunoreactivity was observed in other electron-denseuncharacterized vesicles/organelles (Hatle et al., 2007 Mol Cell Biol27, 2952-2966). To more closely dissect the potential function of MCJ innormal tissue the localization of endogenous MCJ has been examined inheart under physiological conditions by IEM. Most MCJ immunoreactivitywas found in clearly defined mitochondria, predominantly at the innermembrane (FIG. 2A). MCJ expression was also examined in purified CD8 Tcells by IEM. The results showed that MCJ localizes almost exclusivelyat the mitochondria of CD8 T cells (FIG. 2B). A more detailed analysisof MCJ by IEMs in 293T cells overexpressing MCJ for 24 h revealed thepresence of MCJ immunoreactivity in uncharacterized organelles thatcould be undefined, swollen mitochondria that have lost their innermembrane-formed cristae and undergone degradation (FIG. 2C). Confocalmicroscopy analysis for MCJ and Mito Tracker® in 293T cells transfectedwith MCJ (24 h), showed Mito Tracker® staining defining mitochondria innon-transfected cells, while mitochondrial staining was almostundetectable in MCJ-overexpressing cells (FIG. 2 D-F). Thus, MCJ wasfound to localize to the mitochondria under physiological conditions,but its prolonged overexpression in this organelle resulted in apparentmorphological damage.

To further demonstrate the mitochondrial localization of endogenous MCJ,Western blot analysis of MCJ was performed in murine heart mitochondrialand cytosolic fractions. High levels of MCJ were found in themitochondrial fraction while it was almost undetectable in the cytosolicfraction (FIG. 2G). The purity of the fractions was determined by theexpression of Complex IV (CoxIV) of the respiratory chain as a marker ofmitochondria, and GAPDH as a marker of the cytosolic fraction (FIG. 2G).MCJ was also almost exclusively present in the mitochondrial fraction ofpurified CD8 T cells (FIG. 2H). No MCJ could be detected in the nuclearfraction (data not shown). Furthermore, Western blot analysis ofendogenous human MCJ in the breast cancer MCF7 cell line also showed thepresence of MCJ primarily in mitochondria (FIG. 2I). Thus, endogenousMCJ localizes preferentially in mitochondria, and within themitochondria it appears to be anchored to the inner membrane of thecristae.

MCJ Functions as a Negative Regulator of Mitochondrial MembranePotential and ATP Production.

A major function of mitochondria is to provide ATP as a source of energyfor the cell through oxidative phosphorylation. In addition to thetransfer of electrons, Complexes I, III and IV of the mitochondriaelectron transfer chain (ETC) contribute to the establishment of themitochondrial membrane potential (MMP) by promoting the transport of H+across the membrane from the mitochondria matrix to the intermembranespace. Complex V (ATP synthase) uses the energy generated by the flowback of H+ into the mitochondrial matrix to generate ATP. To determinewhether MCJ could modulate mitochondrial function, ATP levels in MCF7breast cancer cells known to express MCJ (Hatle et al., 2007 Mol CellBiol 27, 2952-2966) were compared with the levels in MCF7/siMCJ cells,MCF7 cells where MCJ expression was knocked down by a MCJ shRNA (Hatleet al., 2007 Mol Cell Biol 27, 2952-2966). Interestingly, the levels ofATP in MCF7/siMCJ cells were remarkably higher than in MCF7 cells (FIG.3A). The elevated amounts of ATP in MCF7/siMCJ cells were predominantlyderived from mitochondria because inhibition of the ETC Complex I withrotenone abrogated the levels of ATP (FIG. 3B). These results suggestedthat MCJ is a negative regulator of mitochondrial ATP synthesis. Tofurther confirm these results, the levels of ATP were examined in 293Tcells following expression of MCJ. 293T cells were transfected only for16 h to avoid the mitochondrial damaged observed after 24 h (FIG. 2 ).ATP levels were highly reduced in cells expressing MCJ relative toplasmid control cells (FIG. 3C), further demonstrating that MCJnegatively regulates ATP production.

Because MCJ localizes in the inner membrane of the mitochondria, whetherMCJ could impair MMP as a mechanism for limiting mitochondrial ATPsynthesis was investigated. 293T cells were transfected (16 h) with MCJor a control plasmid, and MMP was examined by TMRE staining and flowcytometry analysis. Expression of MCJ clearly depolarized mitochondria,as determined by the lower TMRE intensity (FIG. 3D), indicating that MCJexpression can dissipate MMP. A prolonged mitochondrial depolarizationis likely the cause of the mitochondria damage observed in 293T cellstransfected for a longer period of time (FIG. 2 ). Because MCJ isabundantly expressed in CD8 compared with CD4 T cells (FIG. 2 ),differences in MMP between the two cell types freshly isolated from wildtype mice were also analyzed. Interestingly, in correlation with theselective presence of MCJ in CD8 T cells, mitochondria were depolarizedin most CD8 T cells compared with mitochondria in CD4 T cells (FIG. 3E).The ETC can also contribute to the generation of mitochondrial reactiveoxygen species (mROS) due to electron escape primarily from Complex III(Hamanaka and Chandel, 2010 Trends Biochem Sci 35, 505-513). Unlike MMP,analysis of mitochondrial ROS (mROS) by staining with MitoSox®-Red andflow cytometry showed no difference in the levels of mROS between CD4and CD8 T cells (FIG. 3F). To further dissect the role of MCJ inmitochondrial function and determine whether the depolarized state ofCD8 T cells is caused by the presence of MCJ, MCJ-deficient mice weregenerated.

The genotype of MCJ deficient mice was confirmed by Southern blotanalysis (FIG. 4A). To confirm the loss of MCJ expression, endogenousMCJ protein levels were examined in different tissues by Western blotanalysis. MCJ was detected in heart and liver from wild type mice butnot in MCJ knockout (KO) mice (FIG. 4B). MCJ expression was alsoabrogated in CD8 T cells from MCJ KO mice (FIG. 4B). No MCJ could bedetected in CD4 T cells from either wild type or MCJ KO mice (FIG. 4B).In addition, no MCJ mRNA could be detected in the MCJ targeted mice,confirming the loss of MCJ expression (FIG. 4C). MCJ mRNA levels werealso reduced in the heterozygous mice compared to wild type CD8 T cells(FIG. 4C), suggesting that MCJ expression in CD8 T cells is dependent onthe allele copy number. Disruption of MCJ expression did not affect theviability of the mice up to the examined age (approximately one year).Both male and female MCJ-deficient mice were fertile and did not exhibitany obvious malformations or behavioral abnormalities (data not shown).Thus, MCJ is not essential for development and/or normal organ functionunder physiological conditions. To demonstrate that MCJ contributes tomaintaining mitochondria in a depolarized state, MMP was examined in CD8T cells isolated from wild type and MCJ KO mice. Relative to wild typeCD8 T cells, CD8 T cells from MCJ KO mice were hyperpolarized to a levelcomparable to wild type CD4 T cells (FIG. 3G). No difference in mROSbetween wild type and MCJ KO CD8 T cells could be observed (FIG. 3H). Asexpected, the analysis of CD4 T cells from wild type and MCJ KO miceshowed no difference in MMP (FIG. 3I) or mROS (FIG. 3J), consistent withthe low expression of MCJ in CD4 T cells.

Together, the results demonstrated that MCJ is a negative regulator ofthe mitochondria electron transfer chain, and the presence of MCJ servesto maintain mitochondria in a depolarized state and restrictmitochondrial generation of ATP. Loss of MCJ therefore leads tomitochondria hyperpolarization.

MCJ Attenuates Mitochondrial Metabolism During the Contraction Phase ofEffector CD8 T Cells.

Whether the hyperpolarization of mitochondria in MCJ KO CD8 T cellsrelative to wild type CD8 T cells could affect their development, wasexamined. The percentage and number of CD8 T cells in lymph nodes (FIG.5A) and spleen (data not shown) was not affected in MCJ knockout mice.Similarly, no difference in thymocyte populations was observed in MCJ KOmice (data not shown). Thus, MCJ does not seem to be essential for CD8 Tcell development or homeostasis in the peripheral immune system. Theeffect that the loss of MCJ may have in the proliferative response ofCD8 T cells was also examined. Purified CD8 T cells were activated withanti-CD3 and anti-CD28 antibodies. Proliferation was measured byincorporation of [³H]-thymidine. No substantial difference was observedin the CD8 T cell proliferative response between MCJ KO and control wildtype mice (FIG. 6A). In addition, despite the presence of MCJ inmitochondria, CD8 T cells from MCJ deficient mice did not undergo celldeath more rapidly during activation (FIG. 5B). The expression ofactivation markers (e.g. CD69, CD44, CD25) between wild type and MCJ KOCD8 T cells during activation was also comparable (data not shown).Thus, MCJ does not seem to be required for the activation or clonalexpansion of CD8 T cells. The regulation of MMP during activation of CD8T cells with anti-CD3 and anti-CD28 Abs was further examined. MMPprogressively increased in wild type CD8 T cells during activation (FIG.6B). Although MMP also slightly increased with activation in MCJ KO CD8T cells and wild type CD4 T cells, the effect was less pronounced due tothe already highly hyperpolarized stage prior to activation (FIG. 6B).After 2 days, MMP levels in wild type CD8 T cells reached similar levelsto those in activated MCJ KO CD8 T cells and wild type CD4 T cells (FIG.4B). mROS levels were also comparable between wild type and MCJ KO CD8 Tcells two days after activation (FIG. 7 ).

During the contraction phase of the immune response, most effector CD8 Tcells die, but those that survive to eventually become memory cellsundergo changes to restore their metabolism to the basal levels found innon-activated naïve CD8 T cells. MMP regulation during the transition ofeffector to resting stage of CD8 T cells was examined. After two days ofactivation with anti-CD3 and anti-CD28 Abs, wild type and MCJ KO CD8 Tcells were washed and incubated in medium alone for 24 h or 48 h. After24 h, both wild type and MCJ KO CD8 T cells remained hyperpolarized,although MMP remained slightly higher in MCJ KO CD8 T cells (FIG. 6C).Interestingly, however, after 48 h a large fraction of wild type CD8 Tcells had already depolarized to levels found in naïve CD8 T cells, butmost MCJ KO CD8 T cells remained hyperpolarized (FIG. 6C). Thus, MCJ wasfound to be required for depolarization of mitochondria during theresting stage of effector CD8 T cells, and restoring MMP to the lowlevels present in naïve cells. The levels of mROS also decreasedovertime, but there was not a marked difference between wild type andMCJ KO CD8 T cells (FIG. 6D). It was also observed that, while wild typeCD8 T cells reduced their size during the resting period to a sizecomparable to naïve cells, MCJ KO CD8 T cells remained larger (data notshown), suggesting that the sustained mitochondrial metabolism inducedby MCJ deficiency leads to a sustained active cell metabolism. Insupport of this sustained active metabolism, ATP levels remained higherin MCJ-deficient than in wild type\ CD8 T cells during the restingperiod (FIG. 6E). To determine whether the effect of MCJ deficiency onmetabolism could affect cell survival in vitro, live cell recovery wasmeasured. Minimal survival of wild type CD8 T cells was found after 2days resting period in vitro, while almost 100% of MCJ KO CD8 T cellsremained alive (FIG. 6F), indicating that MCJ contributes to thecontraction of the CD8 T cell population upon activation.

To further address the role of MCJ in the metabolism of antigen specificCD8 T cells during the transition of effector to resting stage in vivo,MCJ KO mice were crossed with OT-I TCR transgenic mice expressing a TCRthat recognizes ovalbumin (Hogquist et al., 1994 Cell 76, 17-27) togenerate a monoclonal T cell population. OT-I CD8 T cells purified fromwild type and MCJ KO were activated and expanded in vitro. An equalnumber of each cell type was mixed and co-transferred into wild typehost mice. After 15 days, the presence of donor cells in lymph nodes andspleen from the host mice was examined by flow cytometry analysis usingThy1.1 and Thy1.2 markers, as well as size scatter to determine cellsize. Although the percentage recovery of both donors was comparable(data not shown), most wild type donor OT-1 CD8 T cells (Thy1.1+) weresmall as expected, while a significant fraction of the MCJ KO OT-1 CD8 Tcells (Thy1.1+ Thy1.2+) displayed a large size (FIG. 6G). Thus, theresult indicated that MCJ deficiency results in a prolonged activemetabolic state of effector CD8 T cells in vivo in the absence ofantigen, indicating that MCJ contributes to the attenuation themitochondrial metabolism during the transition from the effector to theresting stage of CD8 T cells.

Enhanced Mitochondrial Metabolism by the Loss of MCJ Prevents LiverSteatosis During Starvation.

The role of MCJ attenuating mitochondrial metabolism correlates with thelack of an abnormal phenotype of MCJ deficient mice under basal(physiological) conditions. However, similar to the induction of theresting phase of effector CD8 T cells, MCJ deficiency could have aneffect in situations wherein the metabolic balance is disrupted. Fastingcauses drastic metabolic changes because the lack of sufficient glucosetriggers the hydrolysis of triglycerides stored in the adipose tissue tofree fatty acids (FFA) that are then mobilized into the plasma andtransported to the liver. In the liver, FFA enter mitochondrialβ-oxidation and are used as a source of energy. Because MCJ is highlyexpressed in the liver, it was hypothesized that increased/sustainedmitochondrial metabolism in the absence of MCJ could accelerate FFAβ-oxidation and minimize their accumulation in the liver. The role ofMCJ in fasting metabolism was examined. Histological analysis of liverunder normal (feeding) conditions showed no detectable differencebetween wild type and MCJ deficient mice (FIG. 8A). The livers of wildtype mice fasted for 36 h showed clear signs of steatosis as determinedby the presence of vacuole-enriched cells (FIG. 8A). In contrast,minimal steatotic signs were detected in livers from fasted MCJ KO mice(FIG. 8A). Analysis of lipid accumulation in frozen sections of liver byOil Red O staining further confirmed the presence of high amounts oflipids in fasted wild type mice, while low levels were present in thelivers of MCJ KO mice (FIG. 8B). These results suggested that asustained mitochondrial oxidation of FFA in MCJ KO mice minimize theaccumulation of lipids in the liver. The serum levels of triglyceridesand FFA were also examined. The levels of both triglycerides (FIG. 8C)and FFA (FIG. 8D) were reduced in fasted MCJ KO mice compared with wildtype mice, further demonstrating that there was an acceleratedconsumption of the stored lipids. Consistently, minimal white fat masswas observed to remain in fasted MCJ KO mice (data not shown). Inaddition, although no obvious difference was found in brown fat innormal conditions (feeding) (FIGS. 9A and 9B) between wild type and MCJKO mice, gross analysis (FIG. 10A) and histological analysis (FIG. 10B)of brown fat after fasting revealed almost its complete absence in MCJKO mice. Thus, as a negative regulator of mitochondrial function, theresults indicated that MCJ plays a role in regulating metabolism duringfasting.

Loss of MCI Prevents Lipid Accumulation and Steatosis in the Liver inResponse to Metabolic Changes.

Despite accelerated metabolism identified in mice during fasting, weightloss caused by the fasting in MCJ KO nice was not statisticallydifferent from the weight loss in wild-type mice (FIG. 11A); even theinitial weights were comparable in the two groups (FIG. 11B). Inaddition, the analysis of glucose levels in blood during fasting showedno statistically significant differences between MCJ KO and wild-typemice, either during the initial drop in glucose (12 h) (FIG. 11C) or therecovery (FIG. 11D). These results suggested the presence of mechanismsto balance the accelerated lipid metabolism in the liver in the absenceof MCJ. Increased β-oxidation of free fatty acids (FFA) in the liver bymitochondria could lead to increased levels of ATP (through(3-oxidation), as well as glycerol, resulting from the lipid breakdown.The accumulation of ATP and glycerol can be sensed by the liver as asignal to initiate glycogenesis (an energy-costly process) to store theexcess energy. Analysis of ATP levels in the liver after fasting wasperformed and confirmed increased ATP levels in the livers of MCJ KOmice relative to the livers of wild-type mice (FIG. 11E). Glycogenlevels in the liver were examined by PAS staining. Glycogen was almostundetectable in the livers of fasted wild-type mice, as expected (FIG.11F). However, high levels of glycogen accumulated in the livers offasted MCJ KO mice (FIG. 11F). In addition to the liver, glycogenesiscan occur to a lesser extent in the cortex of kidneys. Accumulation ofglycogen could also be found in some areas of the kidneys in fasted MCJKO mice by PAS staining. Biochemical analysis of glycogen in liverextracts further demonstrated the selective accumulation of glycogen infasted MCJ KO mice (FIG. 11G). No differences were found between thebasal levels of glycogen in the liver in nonfasted wild-type mice andnonfasted MCJ KO mice (FIG. 11G). The accumulation of glycogen insteadof lipids in the livers of fasted MCJ KO mice correlated with anincreased ratio of liver to body weight relative to wild-type mice (FIG.11H). Glycogen is synthesized by glycogen synthase using UDP-glucose asthe substrate. Glycogen synthase levels were upregulated in livers fromfasted MCJ KO mice relative to wild-type mice (FIG. 11I). In contrast,the levels of phosphoenolpyruvate carboxykinase (PEPCK), an essentialgluconeogenic enzyme in the synthesis of glucose from pyruvate, werecomparable in fasted WT and MCJ KO mice (FIG. 11I), suggesting that theglycogen present in the livers of MCJ KO mice was likely generated withglucose resulted from triglyceride hydrolysis.

According to its negative role in mitochondrial respiration, theseresults show that MCJ is an essential regulator of liver metabolismduring fasting and that the absence of MCJ favors lipid degradation andglycogenesis in the liver. To address whether MCJ could also play a rolein regulating metabolism in response to other altered dietaryconditions, its effect in response to a high-cholesterol diet was alsoinvestigated. High cholesterol is a major health problem worldwide.Wild-type and MCJ KO mice were fed a high-cholesterol diet for 4 weeks[see Plavinskaya, T. et al., Pulm Pharmacol Ther. 2013 August;26(4):405-11. doi: 10.1016/j.pupt.2012.10.002. Epub 2012 Oct. 17;Teratani, T. et al., Gastroenterology. 2012 Jan; 142(1):152-164 (Epub2011 Oct. 10)]. High levels of cholesterol accumulated in the livers ofwild-type mice fed the high-cholesterol diet (FIG. 11J). Similar lowlevels of cholesterol were present in livers from wild-type and MCJ KOmice fed a normal diet (FIG. 11K). Thus, results indicated that MCJmodulates the effects caused by a variety of metabolic disorders.

MCJ Deficiency Delays Tumor Growth.

Although mitochondria respiration is the most efficient mechanism togenerate ATP, in some circumstances cells choose glycolysis despite itslower efficiency. Thus, cancer cells generate their ATP from glycolysisrather than through mitochondrial oxidation even under aerobicconditions, which favors tumor growth. This phenomenon is known asWarburg effect and appears to be a metabolic adaptation of cancer cellsdue an impaired mitochondrial oxidative phosphorylation (Cairns et al.,2011 Nat Rev Cancer 11, 85-95; Koppenol et al., 2011 Nat Rev Cancer 11,325-337; Warburg, 1956 J Mol Biol 272, 477-483). Because MCJ is anegative regulator of MMP and ATP, whether MCJ deficiency could have animpact on tumor progression in vivo was addressed. MCJ expression wasexamined in tumors from MMTVPyMT transgenic mice where the middle T (MT)antigen of the polyomavirus is expressed under the control of the MMTV(Mouse Mammary Tumor Virus) promoter/enhancer, which drives expressionspecifically in mammary epithelial cells (Guy et al., 1992 Mol Cell Biol12, 954-961). MCJ was expressed both in normal mammary gland and inmammary tumors, but the levels appeared to be slightly higher in thetumors (FIG. 12A). Analysis of MCJ cellular localization in theMMTV-PyMT tumors by Western blot analysis further confirmed themitochondrial localization of MCJ in mammary tumors (FIG. 12B). Analysisof MCJ in mitochondrial extracts from normal mammary gland and mammarytumors also confirmed an increased expression of MCJ in mitochondriafrom tumors compared with normal mammary gland mitochondria (FIG. 12C).MCJ KO mice were crossed with MMTV-PyMT mice and followed mammary tumordevelopment. Interestingly, although MMTV-PyMT/MCJ KO mice developedtumors, the kinetics were delayed compared with MMTV-PyMT littermates.Consistent with previous studies (Guy et al., 1992 Mol Cell Biol 12,954-961), tumors were clearly detectable by 10-12 weeks of age inMMTV-PyMT littermates, but no obvious tumors were palpable prior to18-20 weeks in MMTV-PyMT/MCJ KO mice. Survival curves were establishedusing the age of the mice when they had to be euthanized due to thelarge size tumors. They showed a significant delay in tumor growth inMMTV-PyMT/MCJ KO mice compared with MMTV-PyMT mice (FIG. 12D).Histological analysis showed no obvious differences between similar sizetumors from MMTV-PyMT and MMTV-PyMT/MCJ KO mice (FIG. 12E). To addressif the delay in tumor growth was associated with a change in the balanceof mitochondrial respiration versus cytosolic glycolysis as sources ofenergy, ATP levels were examined in cytosol and mitochondrial extractsgenerated from wild type and MCJ KO tumors. There was a significantincrease in the relative ratio of mitochondria/cytosolic ATP levels inthe tumors from MCJ KO mice, compared with the ratio in tumors from wildtype mice (FIG. 12F). Thus, the enhanced mitochondrial metabolism foundin the absence of MCJ correlated with an impaired mammary tumor growth.

MCJ is an Endogenous Inhibitor of Complex I in the MitochondrialElectron Transfer Chain.

To investigate the molecular mechanism by which MCJ could regulatemitochondrial membrane potential and finding potential associatingproteins, a phage display screening was performed using the N-terminalregion of MCJ as bait. The results from the screening revealed one ofthe subunits of Complex I (NDUFv1) within the mitochondria ETC as apotential interacting protein with MCJ (data not shown). MammalianComplex I (NADH-ubiquinone oxidoreductase) contains 49 identifiedsubunits, a flavomononucleotide (FMN) group and eight Fe—S clusters(Clason et al., 2009 J Struct Biol 169, 81-88). Although MCJ has notbeen described as an actual subunit of Complex I, it was possible thatMCJ could interact with Complex I and regulate its activity. Todetermine whether MCJ associates with Complex I, Complex I wasimmunoprecipitated from mitochondrial heart extracts of wild type andMCJ KO mice. The presence of MCJ in the immunoprecipitate was examinedby Western blot analysis. A band corresponding to the molecular weightof MCJ was present in the Complex I immunoprecipitate from wild type butnot MCJ KO mice (FIG. 13A). As a control for the immunoprecipitation theexpression of NDUFA9 and NDUFS3, two well characterized subunits ofComplex I (FIG. 13A) was examined. Thus, MCJ associates with Complex Iof the mitochondria.

To determine whether MCJ could be a regulator of Complex I, Complex Iactivity was examined in heart mitochondria extracts from wild type andMCJ deficient mice. Higher Complex I activity was detected in MCJdeficient hearts compared to wild type hearts (FIG. 13B). MCJ deficiencyhowever, did not affect the total amount of Complex I in mitochondria asdetermined by Western blot analysis for NDUFA9 (FIG. 13C). The selectivepresence of MCJ in CD8 T cells relative to CD4 T cells also suggested apotential difference in the activity of Complex I between these celltypes. Analysis of Complex I activity in mitochondria extracts obtainedfrom freshly isolated CD8 and CD4 T cells indeed revealed substantiallylower Complex I activity in CD8 T cells (FIG. 13D). The levels of NDUFA9and NDUFS3 in the mitochondrial extracts were comparable between CD4 andCD8 T cells (FIG. 13E). More importantly, analysis of Complex I activityin MCJ KO CD8 T cells showed increased activity of the complex comparedwith wild type CD8 T cells (FIG. 13D). No difference in the levels ofNDUFA9 or NDUFS3 in mitochondria could be detected between wild type andMCJ KO CD8 T cells (FIG. 13E). Thus, inactivation of Complex I anddepolarization of mitochondria in CD8 T cells relative to CD4 T cells ismediated by the presence of MCJ in CD8 T cells. The effect of MCJdeficiency in Complex I activity was also examined in human MCF7 cells.Despite the comparable levels of Complex I in the mitochondria in bothcell types (FIG. 13F), higher levels of Complex I activity were presentin MCF7/siMCJ cells (lacking MCJ) compared to MCF7 cells (FIG. 13G).Together, these results showed that MCJ is an endogenous negativeregulator of Complex I of the respiratory chain in mitochondria.

Studies set forth herein reveal a novel role of the MCJ/DnaJC15co-chaperone as a negative regulator of Complex I. It has now been shownfor the first time that MCJ/DnaJC15 has a role in attenuatingmitochondrial metabolism and the loss of MCJ alters in vivo metabolicswitches in different tissues. MCJ may therefore be a target to modulatethe energy balance of the cell.

It has now been shown as outlined in part above herein, that MCJphysically associates with Complex I. The association of MCJ withComplex I is likely dynamic and could be a mechanism to rapidly modulatemitochondrial respiration in situations when the mitochondrial metabolicactivity needs to be reduced (e.g. starvation, stress, etc.). Thisinactivation mechanism for Complex I may be play a role in those tissueswhere mitochondria play an essential role as a source of energy, such asthe heart.

Consistent with the inhibitory role of MCJ in Complex I activity, it hasnow been demonstrated that the loss of MCJ results in increasedmitochondrial membrane potential and increased levels of ATP. A relativeincreased mitochondrial metabolism should not have a significant effectunder physiological conditions. In this regard, under physiologicalconditions MCJ deficient mice do not show an altered phenotype. However,an enhanced mitochondrial metabolism in the absence of MCJ could affectmetabolic switches under stress/pathological situations and affect thecourse of the normal response. It has now been demonstrated through theexperiments set forth herein, that MCJ deficiency alters the metabolismunder fasting conditions, and it reduces the accumulation of lipids inthe liver and prevents steatosis. Although not wishing to be bound byany particular mechanism, the reduction in accumulation of lipids in theliver and steatosis prevention may result by the enhancement of lipidβ-oxidation. Correlating with accelerated lipolysis, less content ofwhite fat and only residual brown fat in MCJ deficient mice was observedafter fasting. Thus, in the initial phases of fasting, the enhancedmitochondrial metabolism caused by loss of MCJ can be beneficial.However, because of the rapid consumption of the available “fuel”, it isthought that longer fasting periods could be highly detrimental in theabsence of MCJ. Evolutionary, the acquisition of MCJ in vertebratescould have been an adaptive phenomenon to decelerate mitochondriarespiration by inhibiting Complex I activity in response to insufficientintake of food, and prolong the lipid reserve energy.

Metabolism is also emerging as an important factor that can influencethe survival and/or function of T cells. Oxidation of fatty acids andthe metabolic rate influence survival of memory CD8 T cells (Araki etal., 2009 Nature 460, 108-112; Maines et al., 2009 Science 325, 484-487;Finlay and Cantrell, 2010 Nat Rev Immunol 11, 109-117). In addition, arecent study has shown that IL-15, a well-known survival factor formemory CD8 T cells, promotes mitochondrial oxidative metabolism and ATPproduction in memory cells by enhancing mitochondrial biogenesis (vander Windt et al., 2012 Immunity 36, 68-78). Increase mitochondrial fattyacid oxidation correlates with increased survival and function of memoryCD8 T cells. Here we show that MCJ contributes to the inactivation ofComplex I and, consequently, depolarization of mitochondria in CD8 Tcells. More importantly, it has now been shown that the absence of MCJduring the resting period of effector CD8 T cells sustains an activemitochondrial metabolism in these cells in vitro and in vivo. Thus, MCJmay also regulate memory CD8 T cell function. CD4 and CD8 T cells priorto activation have been historically considered very similar to eachother. Studies presented herein show for the first time a predominantexpression of MCJ in CD8 T cells relative to CD4 T cells, and a drasticmitochondrial depolarization in CD8 T cells maintained by the presenceof MCJ in this these cells. These revealing findings demonstrate a cleardissociation between CD4 and CD8 T cells regarding mitochondrialmetabolism.

Although under physiological conditions most cells use mitochondriarespiration as a mechanism to generate ATP, tumor cells switch toglycolysis as a mechanism to obtain ATP despite being less efficient, aphenomenon known as Warburg effect. Warburg hypothesized that themetabolic switch to glycolysis in cancer cells could be due to damagedmitochondrial respiration, but the actual mechanism remains unclear. Inrecent years the Warburg effect has been broadly reconsidered based onresults from basic and clinical studies (Cairns et al., 2011 Nat RevCancer 11, 85-95; Koppenol et al., 2011 Nat Rev Cancer 11, 325-337;Levine and Puzio-Kuter, 2010 Science 330, 1340-1344). Thus, whileenhanced or prolonged mitochondrial respiration could be beneficial formany cells, it could also interfere with tumor growth. Here it is nowshown that MCJ deficiency delays in vivo mammary tumor growth,correlating with a modified balance towards mitochondrial respiration asa source of energy. A number of in vitro and/or xenograft studies haveaddressed the role of co-chaperones (e.g. Tid1, MRJ, HLAJ1) in cancerand assign them a predominant tumor suppressor function by interferingwith cell invasion, migration, and/or metastasis (Sterrenberg et al.,2011 Cancer Lett 312, 129-142). None of these co-chaperones have beenassociated with changes in the metabolic state of the tumor cells. Thework described herein includes the first study showing the effect of aco-chaperone in vivo tumor progression due to changes in the metabolicstate. It has previously been shown that loss of MCJ is associated withchemoresistance in breast and ovarian cancer (Hatle et al., 2007 MolCell Biol 27, 2952-2966; Strathdee et al., 2005 Gynecol Oncol 97,898-903). Based on findings described herein, it appears possible thatthe enhanced Complex I activity and mitochondria metabolism in theabsence of MCJ could also confer resistance to specific cancer drugs.

Although several embodiments of the present invention have beendescribed and illustrated herein, those of ordinary skill in the artwill readily envision a variety of other means and/or structures forperforming the functions and/or obtaining the results and/or one or moreof the advantages described herein, and each of such variations and/ormodifications is deemed to be within the scope of the present invention.More generally, those skilled in the art will readily appreciate thatall parameters, dimensions, materials, and configurations describedherein are meant to be exemplary and that the actual parameters,dimensions, materials, and/or configurations will depend upon thespecific application or applications for which the teachings of thepresent invention is/are used. Those skilled in the art will recognize,or be able to ascertain using no more than routine experimentation, manyequivalents to the specific embodiments of the invention describedherein. It is, therefore, to be understood that the foregoingembodiments are presented by way of example only and that, within thescope of the appended claims and equivalents thereto; the invention maybe practiced otherwise than as specifically described and claimed. Thepresent invention is directed to each individual feature, system,article, material, and/or method described herein. In addition, anycombination of two or more such features, systems, articles, materials,and/or methods, if such features, systems, articles, materials, and/ormethods are not mutually inconsistent, is included within the scope ofthe present invention.

All definitions, as defined and used herein, should be understood tocontrol over dictionary definitions, definitions in documentsincorporated by reference, and/or ordinary meanings of the definedterms.

The indefinite articles “a” and “an,” as used herein in thespecification and in the claims, unless clearly indicated to thecontrary, should be understood to mean “at least one.”

The phrase “and/or,” as used herein in the specification and in theclaims, should be understood to mean “either or both” of the elements soconjoined, i.e., elements that are conjunctively present in some casesand disjunctively present in other cases. Other elements may optionallybe present other than the elements specifically identified by the“and/or” clause, whether related or unrelated to those elementsspecifically identified, unless clearly indicated to the contrary.

All references, patents and patent applications and publications thatare cited or referred to in this application are incorporated herein intheir entirety herein by reference.

The invention claimed is:
 1. A method of increasing mitochondrial metabolism in a kidney cell, the method comprising contacting the kidney cell with an MCJ-modulating compound comprising an MCJ siRNA that decreases MCJ polypeptide activity in the kidney cell, in an amount effective to increase mitochondrial metabolism in the kidney cell.
 2. The method of claim 1, wherein decreasing the MCJ polypeptide activity comprises decreasing a level or function of MCJ polypeptide in the kidney cell.
 3. The method of claim 1, wherein mitochondrial metabolism comprises mitochondrial respiration.
 4. The method of claim 1, wherein the increased mitochondrial metabolism reduces lipid accumulation in the kidney cell.
 5. The method of claim 1, wherein the MCJ-modulating compound further comprises a targeting agent. 